Cloning and uses of the genetic locus BCL-6

ABSTRACT

This invention provides a method for detecting a rearrangement of a bcl-6 gene in B-cell lymphoma in a subject, comprising: (a) obtaining a DNA sample from the subject; (b) cleaving the DNA sample into fragments; (c) separating the DNA fragments by size fractionation; (d) hybridizing the DNA fragments with a nucleic acid molecule comprising at least 15 contiguous nucleotides which is complementary to a sequence of an isolated nucleic acid molecule having the nucleic acid sequence as set forth in SEQ ID NO:1 and specifically hybridizes with the nucleic acid sequence as set forth in SEQ ID NO:1 to detect the DNA fragment containing the bcl-6 nucleic acid sequence; and (e) comparing the detected DNA fragment from (d) with a DNA fragment from a known normal subject, the difference in size of the fragments indicating occurrence of a rearrangement of the bcl-6 gene in B-cell lymphoma in the subject. This invention also provides a method for detecting a rearrangement of a bcl-6 gene in B-cell lymphoma in a subject, comprising: (a) obtaining an RNA sample from the subject; (b) separating the RNA sample into different species by size fractionation; (c) hybridizing the RNA species with a nucleic acid molecule comprising at least 15 contiguous nucleotides which is complementary to a sequence of an isolated nucleic acid molecule having the nucleic acid sequence as set forth in SEQ ID NO:1 and specifically hybridizes with the nucleic acid sequence as set forth in SEQ ID NO:1 to detect the RNA species containing bcl-6 nucleic acid sequence; and (d) comparing the detected RNA species from step (c) with the RNA species from a known normal subject, the difference in size of the species indicating the rearrangement of the bcl-6 gene in B-cell lymphoma in the subject.

The invention disclosed herein was made with Government support underNIH Grant Nos. CA-44029, CA-34775, CA-08748 and CA-37295 from theDepartment of Health and Human Services. Accordingly, the U.S.Government has certain rights in this invention.

This application is a continuation-in-part U.S. application Ser. No.08/074,967, filed on Jun. 9, 1993 and issued as U.S. Pat. No. 5,641,672on Jun. 24, 1997, the contents of which are hereby incorporated byreference.

BACKGROUND OF THE INVENTION

Throughout this application various references are referred to withinparenthesis. Disclosures of these publications in their entireties arehereby incorporated by reference into this application to more fullydescribe the state of the art to which this invention pertains. Fullbibliographic citation for these references may be found at the end ofeach Experimental Detail Section.

Non-random chromosomal abnormalities are found in up to 90% of patientswith non-Hodgkin's lymphoma (NHL) and have been shown to play animportant role in lymphomagenesis by activating proto-oncogenes (1).Some of these translocations, which are associated with specifichistologic subsets of NHL, have been characterized at the molecularlevel. In the t(8;14), t(8;22), and t(2;8) translocations associatedwith Burkitt Lymphoma, L₃ -type acute lymphoblastic leukemia andAIDS-associated non-Hodgkin lymphoma (NHL), a known proto-oncogene,c-myc, was found juxtaposed to the immunoglobulin (Ig) loci (2,3). Inthe t(14;18) translocation, which is implicated in follicular-type NHL,molecular analysis of the sequences linked to the Ig locus led to theidentification of a novel proto-oncogene, bcl-2 (4-6). The t(11;14)(q13;q32), mainly associated with "mantle zone" lymphoma, appears toinvolve the juxtaposition of the Ig heavy-chain locus with the bcl-1locus, the site of the candidate proto-oncogene PRAD-1/cyclin D1 (7,8).These well characterized chromosome translocations are associated,however, with only a fraction of NHL cases, while a number of otherrecurrent translocations remain to be characterized for their geneticcomponents.

One important example of such cytogenetic abnormalities is representedby various alterations affecting band 3q27. This region is involved intranslocations with various chromosomal sites including, but notlimited, to those carrying the Ig heavy- (14q32) or light- (2p12, 22q11)chain loci (9,10). Overall, 3q27 breakpoints are detectable in 7-12% ofB-cell NHL cases by cytogenetic analysis, with t(3;22) (q27;q11) beingthe most frequent type detectable in 4-5% of NHL (9). Theclinicopathologic relevance of 3q27 breakpoints is underscored by itsconsistent association with diffuse-type NHL, a frequent and clinicalaggressive subtype for which no specific molecular lesion has yet beenidentified (9).

The recurrence of 3q27 breakpoints in NHL has prompted a search for thecorresponding proto-oncogene. This invention discloses the cloning ofclustered 3q27 breakpoints from two NHL cases carrying t(3;14) (q27;q32)translocations and the identification of genomic rearrangements withinthe same breakpoint region in additional NHL cases carryingtranslocations involving 3q27. Within the same region, a transcriptionalunit has been identified, which represents the candidate proto-oncogene(bcl-6) associated with 3q27 translocations in B-NHL.

SUMMARY OF THE INVENTION

This invention provides an isolated vertebrate nucleic acid molecule ofbcl-6 locus. This invention provides an isolated vertebrate DNA moleculeof bcl-6 locus. This invention provides an isolated vertebrate cDNAmolecule of bcl-6. This invention provides an isolated genomic DNAmolecule of bcl-6. This invention provides an isolated vertebrate RNAmolecule of bcl-6. This invention provides an isolated human nucleicacid molecule of bcl-6 locus.

In addition, this invention provides a nucleic acid molecule comprisinga nucleic acid molecule of at least 15 nucleotides capable ofspecifically hybridizing with a sequence included within the sequence ofthe nucleic acid molecule of bcl-6.

In addition, this invention provides an isolated vertebrate DNA moleculeof bcl-6 operatively linked to a promoter of RNA transcription. Thisinvention provides a vector which comprises the isolated vertebrate DNAmolecule of bcl-6.

In addition, this invention provides the above vector, wherein theisolated nucleic acid molecule is linked to a plasmid.

In addition, this invention provides a host vector system for theproduction of a polypeptide encoded by bcl-6 locus, which comprises theabove vector in a suitable host.

In addition, this invention provides a method of producing a polypeptideencoded by bcl-6 locus, which comprises growing the above host vectorsystem under suitable conditions permitting production of thepolypeptide and recovering the polypeptide so produced.

In addition, this invention provides a polypeptide encoded by theisolated vertebrate nucleic acid molecule of bcl-6 locus. Further, thisinvention provides an antibody capable of binding to polypeptide encodedby bcl-6 locus.

In addition, this invention provides an antagonist capable of blockingthe expression of the polypeptide encoded by bcl-6.

In addition, this invention provides an antisense molecule capable ofhybridizing to the nucleic acid molecule of bcl-6.

In addition, this invention provides an assay for non-Hodgkin'slymphoma, a method for screening putative therapeutic agents fortreatment of non-Hodgkin's lymphoma and a method for diagnosing B-celllymphoma.

Finally, this invention provides a method of treating a subject withnon-Hodgkin's lymphoma.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Immunoglobulin gene rearrangement analysis of KC1445 and SM1444DNA. DNA extracted from the cell lines U937 (monocytic leukemia) andSK-N-MC (neuroblastoma) were used as controls for non-rearranged,germ-line Ig genes. In the left panel, the arrow on the left points tothe rearranged J_(H) fragment which does not contain C.sub.μ sequencesin KC1445 DNA, while the two arrows on the right point to the twodistinct fragments containing J_(H) or C.sub.μ sequences in SM1444 DNA.

FIG. 2: Molecular cloning of the chromosomal breakpoints from two NHLcases with t(3;14). Illustrated are the maps of two representative phageclones spanning the breakpoint regions in case SM1444 (SM-71) and KC1445(KC-51). Chromosome 14 portions of the phage inserts are indicated by asolid line with hatched and black boxes representing switch sequencesand C.sub.μ exons, respectively. Vertical arrows point to the junctionsof chromosome 3 and 14 sequences. The probes used for Southern (FIG. 4)and Northern (FIG. 5) analysis are illustrated below the SM-71 map.Restriction enzyme sites are indicated as: B=BamHI; H=HindIII; R=EcoRI;G=BblII; S=sacI.

FIGS. 3A-3B: Localization of phage SM-71 sequences to chromosomes 3 and14 by fluorescence in situ hybridization. Consistent hybridizationsignals at 3q27 (arrow in panel A) and 14q32 (arrow in panel B)demonstrated that the insert is derived from the translocation junction.

FIGS. 4A-4C: Southern blot hybridization analysis of bcl-6rearrangements in NHL carrying 3q27 breakpoints. The probes used areillustrated in FIG. 2. U937 and SK-N-MC DNAs are used as germ-linecontrols since their hybridization pattern was identical to the oneobserved in a panel of 19 control DNAs tested. The detected cytogeneticabnormalities affecting 3q27 in each case are: KC1445: t(3;14)(q27;q32); SM1444: t(3;14) (q27;q32); TF1403: t(3;14) (q27;q32); LD1411:t(3,14) (q27;q32); EM352: t(3;22) (q27;q11); CF755: t(3;12) (q27;q11);S0955: der(3)t(3;5) (q27;q31).

FIG. 5: Identification of the bcl-6 transcriptional unit. 15 μg of totalRNA isolated from the indicated human cell lines was analyzed byNorthern blot hybridization using the Sac 4.0 probe (see FIG. 2).CB33:EBV-immortalized human B lymphoblastoid cell line; HeLa: humancervical carcinoma cell line; Daudi: human Burkitt lymphoma cell line;Hut78: human T-cell leukemia cell line. Hybridization of the same filterto a mouse GAPDH probe is shown as control for RNA amount loaded in eachlane. The faint band comigrating with 28S RNA in all the lanes may bethe result of cross-hybridization with ribosomal RNA sequences.

FIG. 6: Map of normal human BCL-6 locus. A recombinant genomic DNAlibrary derived from normal placenta DNA was obtained from STRATAGENEInc and screened by plaque hybridization using the Sac 4.0 probe. Threerecombinant phages were obtained (φ1-3 in the figure) whose inserts havebeen mapped and shown to overlap on approximately 30 kilobases ofgenomic DNA representing the BCL-6 locus. These sequences containingbcl-6 exons since they hybridize to the cDNA probe. The precise positionof the exons has only been approximately determined and is schematicallyindicated in the figure. The position of the breakpoints observed invarious lymphoma cases is also indicated.

FIG. 7: psac 40 plasmid construction.

FIG. 8: pGB31 and pGB3s plasmid construction.

FIGS. 9A-9D: cDNA and Amino Acid Sequences of BCL-6 (SEQ ID NOs. 1 and2). The Sac 4.0 probe was used to screen a recombinant phage cDNAlibrary constructed from Bjab B cell lymphoma line RNA. A 4.0 kilobasecDNA was isolated and its nucleotide sequence was determined. Itcontains a long open reading frame potentially coding for 706 amino acidprotein which contains five zinc-finger domains (underlined in thefigure; C and H residues which identify the C2H2-type zinc-fingerstructure are indicated in bold).

FIGS. 10A-10B: Structure of BCL-6 cDNA and sequence of its predictedprotein product. FIG. 10A: Schematic representation of the full-lengthBCL-6 cDNA clone showing the relative position of the open reading frame(box) with 5' and 3' untranslated sequences (lines flanking the box).The approximate positions of the zinc-finger motifs (Zn++) and the NH₂-terminal homology (shaded area) with other proteins are also indicated.FIG. 10B SEQ ID NO: 2: The predicted amino acid sequence of the BCL-6protein. The residues corresponding to the six zinc-finger motifs (H-Clinks). The GenBank Accession number for BCL-6 cDNA and amino acidsequences is U00115.

FIG. 11: Homology of the NH₂ -terminal region of BCL-6 to other Kruppelzinc-finger proteins, viral (VA55R), or cellular non-zinc-finger (kelch)proteins SEQ ID NO: 3-9. Black background indicates identical residuesfound four or more times at a given position; grey indicates conservedresidues that appear in at least four sequences at a given position.Conserved amino acid substitutions are defined according to scheme (P,A, G, S, T), (Q, N, E, D), (H, K, R), (L, I, V, M), and (F< Y< W)Numbering is with respect to the methionine initiation codon of eachgene.

FIG. 12: Exon-intron organization of the BCL-6 gene and mapping ofbreakpoints detected in DLCL. Coding and non-coding exons arerepresented by filled and empty boxes, respectively. The position andsize of each exon are approximate and have been determined by thepattern of hybridization of various cDNA probes as well as by thepresent of shared restriction sites in the genomic and cDNA. Theputative first, second and third exons have been sequenced in theportions overlapping the cloned cDNA sequences. The transcriptioninitiation site has not been mapped (shaded box on 5' side of firstexon). Patient codes (e.e. NC11, 891546 etc.) are grouped according tothe rearranged patterns displayed by tumor samples. Arrows indicate thebreakpoint position for each sample as determined by restrictionenzyme/hybridization analysis. For samples KC1445 and SM1444, thebreakpoints have been cloned and the precise positions are known.Restriction sites marked by asterisks have been only partially mappedwithin the BCL-6 locus. Restriction enzyme symbols are: S, Sac I; B, BamHI; X, Xba I; H, Hind III, R, Eco RI; G, Bgl II; P, Pst I; Sc, Sca I;St, Stu I; Rs, Rsa I. Tumor samples were collected and analyzed forhistopathology at Memorial Sloan-Kettering Cancer Center or at ColumbiaUniversity.

FIGS. 13A-13B: Rearrangements of the BCL-6 gene in diffuse large-celllymphomas (DLCL). Genomic DNA extracted from tumor biopsies of DLCLcases and from normal lymphocytes (lane N) was digested with theindicated restriction enzymes and analyzed by Southern blothybridization using the Sac 4.0 probe. Abnormal restriction fragmentsare indicated by the arrows.

FIGS. 14A-14C: Analysis of BCL-6 rearrangements in AIDS-NHL (FIGS.14A-14C). DNAs were digested with BamHI (FIG. 14A) or XbaI (FIGS. 14Band 14C) and hybridized to probes Sac4.0 (FIGS. 14A and 14B) or Sac0.8(FIG. 14C). The BCL-6 germline bands detected by BamHI (11.4 Kb) andXbaI (14 Kb) are indicated. U937 was used as a BCL-6 germline control.Among the cases shown, rearrangements were detected in cases DK782,DK827, and DS16, represented by AIDS-DLCL.

FIG. 15: Restriction map of the germline BCL-6 locus. Exon-intronorganization of the BCL-6 gene. Coding and noncoding exons arerepresented by filled and empty boxes, respectively. The transcriptioninitiation site has not been mapped (shaded box on 5' side of firstexon). The breakpoints detected in AIDS-NHL are indicated by arrows.Restriction enzyme symbols are: S, SacI; B, BamHI; X, XbaI, R, EcoRI.RE, restriction enzyme.

FIGS. 16A-16C: Analysis of EBV infection (FIG. 16A), c-MYCrearrangements (FIG. 16B), and p53 mutations (FIG. 16C) in AIDS-NHL.FIG. 16A: Analysis of EBV termini heterogeneity in AIDS-NHL. DNAs weredigested with BamHI and subjected to Southern hybridization using a DNAprobe specific for the fused termini of the EBV genome. U937, amonocytic leukemia cell line, is used as a negative control. Alymphoblastoid cell line derived by EBV infection of normal polyclonal Bcells (NC2) is used as control for polymorphic EBV termini.Representative samples of AIDS-NHL, both positive (DK3794, DK4338,DK2814, DK3973) and negative (DK3479), are shown. FIG. 16B: Southernblot analysis of c-MYC rearrangements in AIDS-NHL. Genomic DNAs from thecases shown was digested with HindIII and probed with clone MC413RC⁴¹,representative of c-MYC exon 3. A lymphoblastoid cell line (NC2) wasused as control for c-MYC germline configuration. Among the cases shown,two cases of AIDS-DLCL (DK3537 and DK1446) display a c-MYCrearrangement. FIG. 16C: Analysis by PCR-SSCP of the p53 gene inAIDS-NHL. Representative examples are shown for p53 exon 5. Samples werescored as abnormal when differing from the normal control (N). A sampleknown to harbor a p53 mutation was used as positive control (POS). Amongthe cases shown, DK1171, a case of AIDS-SNCCL, shows a p53 mutationwhich was further characterized by direct sequencing of the PCR product.

FIGS. 17A-17B: Southern blot analysis of the BCL-6 gene configuration indiffuse large cell lymphomas. Genomic DNA extracted from tumor biopsieswas digested with the indicated restriction endonucleases and hybridizedusing the Sac4.0 probe (19). Rearranged fragments are indicated by thearrows. N=normal control DNA obtained from human lymphocytes.

FIGS. 18A-18B: FIG. 18A: Freedom from progression in BCL-6 rearrangedcases (open circles, top curve) compared to BCL-6 germline cases (closedcircles, bottom curve) (P=0.007). FIG. 18B: Overall survival from timeof diagnosis for BCL-6 rearranged CLLC (open circle, top curve),compared to BCL-6 germline, BCL-2 germline DLLC (dark triangles, middlecurve), and BCL-2 rearranged DLLC (dark boxes, bottom curve) (P=0.02).

DETAILED DESCRIPTION OF THE INVENTION

The following standard abbreviations are used throughout thespecification to indicate specific nucleotides:

    ______________________________________    C = cytosine        A = adenosine    T = thymidine       G = guanosine    ______________________________________

This invention provides an isolated vertebrate nucleic acid molecule ofthe bcl-6 locus. As used herein, bcl-6 locus means the breakpointcluster region in B-cell lymphomas. The bcl-6 locus is of 30 kilobase inlength containing at least a bcl-6 gene which codes for a protein.Therefore, the bcl-6 locus contains both the 5' and 3' flanking regionof the coding sequences of the bcl-6 gene.

In an embodiment, the isolated, vertebrate nucleic acid molecule ofbcl-6 locus is DNA. In another embodiment, the isolated, vertebratenucleic acid of the bcl-6 locus is cDNA. In a further embodiment, theisolated, vertebrate nucleic acid is genomic DNA. In a still furtherembodiment, the isolated, vertebrate nucleic acid molecule is RNA.

This invention provides an isolated, human nucleic acid moleculecomprising the bcl-6 locus.

The DNA molecules described and claimed herein are useful for theinformation which they provide concerning the amino acid sequence of thepolypeptide and as products for the large scale synthesis of thepolypeptide by a variety of recombinant techniques. The molecule isuseful for generating new cloning and expression vectors, transformedand transfected prokaryotic and eukaryotic host cells, and new anduseful methods for cultured growth of such host cells capable ofexpression of the polypeptide and related products.

Moreover, the isolated vertebrate nucleic acid molecules are useful forthe development of probes to study B cell lymphomas.

This invention provides a nucleic acid molecule comprising a nucleicacid molecule of at least 15 nucleotides capable of specificallyhybridizing with a sequence included within the sequence of the bcl-6locus. In an embodiment, this molecule is DNA. In another embodiment,the molecule is RNA.

As used herein, the phrase "specifically hybridizing" means the abilityof a nucleic acid molecule to recognize a nucleic acid sequencecomplementary to its own and to form double-helical segments throughhydrogen bonding between complementary base pairs.

The above nucleic acid molecule of at least 15 nucleotides capable ofspecifically hybridizing with a sequence of bcl-6 locus may be used as aprobe for bcl-6 sequences. Nucleic acid probe technology is well knownto those skilled in the art who will readily appreciate that such probesmay vary greatly in length and may be labeled with a detectable label,such as a radioisotope or fluorescent dye, to facilitate detection ofthe probe. DNA probe molecules may be produced by insertion of a DNAmolecule having the full-length or a fragment of the bcl-6 locus intosuitable vectors, such as plasmids or bacteriophages, followed bytransforming into suitable bacterial host cells, replication in thetransformed bacterial host cells and harvesting of the DNA probes, usingmethods well known in the art. Alternatively, probes may be generatedchemically from DNA synthesizers.

RNA probes may be generated by inserting the full length or a fragmentof the bcl-6 locus downstream of a bacteriophage promoter such as T3, T7or SP6. Large amounts of RNA probe may be produced by incubating thelabeled nucleotides with a linearized bcl-6 or its fragment where itcontains an upstream promoter in the presence of the appropriate RNApolymerase.

This invention provides an cDNA molecule of bcl-6 locus operativelylinked to a promoter of RNA transcription.

This invention provides a vector which comprises the nucleic acidmolecule of bcl-6 locus. This invention provides the above vector,wherein the isolated nucleic acid molecule is linked to a plasmid.

This invention further provides isolated cDNA molecule of the bcl-6locus operatively linked to a promoter of RNA transcription. Variousvectors including plasmid vectors, cosmid vectors, bacteriophage vectorsand other viruses are well known to ordinary skilled practitioners.

As an example to obtain these vectors, insert and vector DNA can both beexposed to a restriction enzyme to create complementary ends on bothmolecules which base pair with each other and are then ligated togetherwith DNA ligase. Alternatively, linkers can be ligated to the insert DNAwhich correspond to a restriction site in the vector DNA, which is thendigested with the restriction enzyme which cuts at that site. Othermeans are also available and known to an ordinary skilled practitioner.

In an embodiment, a partial CDNA molecule of the bcl-6 locus is linkedto pGEM-7zf(-) and the resulting plasmid is designated as pGB31 (FIG.8). Plasmid, pGB31 was deposited on Jun. 3, 1993 with the American TypeCulture Collection (ATCC), 12301 Parklawn Drive, Rockville, Md. 20852,U.S.A. under the provisions of the Budapest Treaty for the InternationalRecognition of the Deposit of Microorganism for the Purposes of PatentProcedure. Plasmid, pGB31 was accorded with ATCC Accession Number 75476.

In an another embodiment, a partial cDNA molecule of the bcl-6 locus islinked to pGEM-7zf(-) and the resulting plasmid is designated as pGB3s(FIG. 8). Plasmid, pGB3s was deposited on Jun. 3, 1993 with the AmericanType Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Md.20852, U.S.A. under the provisions of the Budapest Treaty for theInternational Recognition of the Deposit of Microorganism for thePurposes of Patent Procedure. Plasmid, pGB3s was accorded with ATCCAccession Number 75477.

This invention provides a host vector system for the production of apolypeptide encoded by bcl-6 locus, which comprises the above vector ina suitable host.

This invention provides the above host vector system, wherein thesuitable host is a bacterial cell, insect cell, or animal cell.

Regulatory elements required for expression include promoter sequencesto bind RNA polymerase and transcription initiation sequences forribosome binding. For example, a bacterial expression vector includes apromoter such as the lac promoter and for transcription initiation theShine-Dalgarno sequence and the start codon AUG. Similarly, a eukaryoticexpression vector includes a heterologous or homologous promoter for RNApolymerase II, a downstream polyadenylation signal, the start codon AUG,and a termination codon for detachment of the ribosome. Such vectors maybe obtained commercially or assembled from the sequences described bymethods well-known in the art, for example the methods described abovefor constructing vectors in general. Expression vectors are useful toproduce cells that express the polypeptide encoded by the bcl-6 locus.

This invention further provides an isolated DNA or cDNA moleculedescribed hereinabove wherein the host cell is selected from the groupconsisting of bacterial cells (such as E. coli), yeast cells, fungalcells, insect cells and animal cells. Suitable animal cells include, butare not limited to Vero cells, HeLa cells, Cos cells, CV1 cells andvarious primary mammalian cells.

This invention provides a method of producing a polypeptide encoded bybcl-6 locus, which comprises growing the above host vector system undersuitable conditions permitting production of the polypeptide andrecovering the polypeptide so produced.

This invention provides a polypeptide encoded by the isolated vertebratenucleic acid molecule of bcl-6 locus.

This invention provides an antibody capable of binding to polypeptideencoded by bcl-6 locus. In an embodiment, the antibody is monoclonal.

This invention provides a method to select specific regions on thepolypeptide encoded by the bcl-6 locus to generate antibodies. Theprotein sequence may be determined from the cDNA sequence. Amino acidsequences may be analyzed by methods well known to those skilled in theart to determine whether they produce hydrophobic or hydrophilic regionsin the proteins which they build. In the case of cell membrane proteins,hydrophobic regions are well known to form the part of the protein thatis inserted into the lipid bilayer of the cell membrane, whilehydrophilic regions are located on the cell surface, in an aqueousenvironment. Usually, the hydrophilic regions will be more immunogenicthan the hydrophobic regions. Therefore the hydrophilic amino acidsequences may be selected and used to generate antibodies specific topolypeptide encoded by the bcl-6 locus. The selected peptides may beprepared using commercially available machines. As an alternative, DNA,such as a cDNA or a fragment thereof, may be cloned and expressed andthe resulting polypeptide recovered and used as an immunogen.

Polyclonal antibodies against these peptides may be produced byimmunizing animals using the selected peptides. Monoclonal antibodiesare prepared using hybridoma technology by fusing antibody producing Bcells from immunized animals with myeloma cells and selecting theresulting hybridoma cell line producing the desired antibody.Alternatively, monoclonal antibodies may be produced by in vitrotechniques known to a person of ordinary skill in the art. Theseantibodies are useful to detect the expression of polypeptide encoded bythe bcl-6 locus in living animals, in humans, or in biological tissuesor fluids isolated from animals or humans.

The antibody may be labelled with a detectable marker, including but notlimited to: a radioactive label, or a calorimetric, luminescent, orfluorescent marker, or gold. Radioactive labels include but are notlimited to: ³ H, ¹⁴ C, ³² P, ³³ P; ³⁵ S, ³⁶ Cl, ⁵¹ Cr, ⁵⁷ Co, ⁵⁹ Co, ⁵⁹Fe, ⁹⁰ Y, ¹²⁵ T, ¹³¹ I, and ¹⁸⁶ Re. Fluorescent markers include but arenot limited to: fluorescein, rhodamine and auramine. Methods ofproducing the polyclonal or monoclonal antibody are known to one ofordinary skill in the art.

Further, the antibody complex may be detected by a second antibody whichmay be linked to an enzyme, such as alkaline phosphatase or horseradishperoxidase. Other enzymes which may be employed are well known to one ofordinary skill in the art.

This invention provides for the isolated nucleic acid molecule of bcl-6that is labelled with a detectable marker. The detectable marker may bea radioactive label, a calorimetric, luminescent, or a fluorescentmarker. Other detectable markers are known to those skilled in the artas hereinabove described.

This invention provides an antagonist capable of blocking the expressionof the polypeptide encoded by the isolated nucleic acid molecule ofbcl-6. The antagonist may be a triplex oligonucleotide capable ofhybridizing to nucleic acid molecule bcl-6.

This invention provides an antisense molecule capable of hybridizing tothe nucleic acid molecule bcl-6. The antisense molecule may be DNA orRNA.

This invention provides a triplex oligonucleotide capable of hybridizingwith a double stranded DNA molecule bcl-6.

The antisense molecule may be DNA or RNA or variants thereof (i.e. DNAwith a protein backbone). The present invention extends to thepreparation of antisense nucleotides and ribozymes that may be used tointerfere with the expression of the receptor recognition proteins atthe translation of a specific mRNA, either by masking that mRNA with anantisense nucleic acid or cleaving it with a ribozyme.

Antisense nucleic acids are DNA or RNA molecules that are complementaryto at least a portion of a specific mRNA molecule. In the cell, theyhybridize to that mRNA, forming a double stranded molecule. The celldoes not translate an MRNA in this double-stranded form. Therefore,antisense nucleic acids interfere with the expression of mRNA intoprotein. Oligomers of about fifteen nucleotides and molecules thathybridize to the AUG initiation codon will be particularly efficient,since they are easy to synthesize and are likely to pose fewer problemsthan larger molecules upon introduction to cells.

This invention provides a transgenic nonhuman mammal which comprises theisolated nucleic acid molecule bcl-6 introduced into the mammal at anembryonic stage.

This invention provides an assay for non-Hodgkin's lymphoma, comprising(a) incubating a sample of suitable body fluid for a subject with amonoclonal antibody reactive with non-Hodgkin's lymphoma cells to asolid support, (b) removing unbound body fluid from the support, and (c)determining the level of antigen activity exhibited by the bound bodyfluid to the support.

The suitable bodily fluid sample is any bodily fluid sample which wouldcontain non-hodgkin lymphoma cells or fragments thereof. A suitablebodily fluid includes, but is not limited to, serum, plasma,cerebrospinal fluid, and urine. In the preferred embodiment, thesuitable bodily fluid sample is serum or plasma. In addition, the bodyfluid sample may cells from bone marrow, or a supernate from a cellculture. Methods of obtaining a suitable bodily fluid sample from asubject are known to those skilled in the art.

This invention provides a method for screening putative therapeuticagents for treatment of non-Hodgkin's lymphoma, which comprisesdetermining in a first sample from a subject with non-Hodgkin's lymphomathe presence of the isolated nucleic acid molecule bcl-6, administeringto the subject a therapeutic amount of the agent such that the agent iscontacted with the cell associated with the condition, determining aftera suitable period the amount of the isolated nucleic acid molecule in asample from the treated subject, and comparing the amount of isolatednucleic acid molecule determined in the first sample with the amountdetermined in the sample from the treated subject, a differenceindicating the effectiveness of the agent, thereby screening putativetherapeutic agents for treatment of non-Hodgkin's lymphoma.

Further, this invention provides an assay system that is employed toidentify drugs or other molecules capable of binding to the nucleic acidmolecule bcl-6 or proteins, either in the cytoplasm or in the nucleus,thereby inhibiting or potentiating transcriptional activity. Such assaywould be useful in the development of drugs that would be specificagainst particular cellular activity, or that would potentiate suchactivity, in time or in level of activity.

The above described probes are also useful for in-situ hybridization orin order to locate tissues which express this gene, or for otherhybridization assays for the presence of this gene or its mRNA invarious biological tissues.

The in-situ hybridization technique using the labelled nucleic acidmolecule bcl-6 is well known in the art. Essentially, tissue sectionsare incubated with the labelled nucleic acid molecule to allow thehybridization to occur. The molecule will carry a marker for thedetection because it is "labelled", the amount of the hybrid will bedetermined based on the detection of the amount of the marker. Further,immunohistochemical protocols may be employed which are known to thoseskilled in the art.

This invention provides a method of diagnosing diffuse-type B-celllymphoma in a subject which comprises detecting in a sample from thesubject nucleic acid molecule of bcl-6 locus.

This invention provides a method for diagnosing B-cell lymphoma in asubject comprising: (a) obtaining DNA sample from the subject; (b)cleave the DNA sample into fragments; (c) separating the DNA fragmentsby size fractionation; (d) hybridizing the DNA fragments with a nucleicacid molecule comprising a nucleic acid molecule of at least 15nucleotides capable of specifically hybridizing with a sequence includedwithin the sequence of the nucleic acid molecule of the bcl-6 locus todetect the DNA fragment containing the bcl-6 sequence; and (e) comparingthe detected DNA fragment from step (d) with the DNA fragment from aknown normal subject, the difference in size of the fragments indicatingthe occurrence of B-cell lymphoma in the subject. In a preferredembodiment, the above diagnostic method is for diffuse-type B-celllymphomas.

A person of ordinary skill in the art will be able to obtain appropriateDNA sample for diagnosing B-cell lymphoma in a subject. The DNA sampleobtained by the above described method may be cleaved by restrictionenzyme. The uses of restriction enzymes to cleave DNA and the conditionsto perform such cleavage are well-known in the art.

In an embodiment, the size fractionation in step (c) of theabove-described method is effected by a polyacrylamide gel. In anotherembodiment, the size fractionation is effected by an agarose gel.

This invention also provides the above-described diagnosis methodwherein step the nucleic acid molecule in step (d) is labeled with adetectable marker. The detectable marker includes but is not limited toa radiolabelled molecule, a fluorescent molecule, an enzyme, or aligand.

In a preferred embodiment, the above-described diagnosis method furthercomprises transferring the DNA fragments into a solid matrix before thehybridization step (d). One example of such solid matrix isnitrocellulose paper.

As an example for the above-described diagnosis method is shown in FIGS.4A-4C where different NHL sample are analyzed. More lymphoma cases andtheir breakpoints are shown in FIG. 6.

This invention also provides a method for diagnosing B-cell lymphoma ina subject comprising: (a) obtaining RNA sample from the subject; (b)separating the RNA sample into different species by size fractionation;(c) hybridizing the RNA species with a nucleic acid molecule comprisinga nucleic acid molecule of at least 15 nucleotides capable ofspecifically hybridizing with a sequence included within the sequence ofthe nucleic acid molecule of the bcl-6 locus to detect the RNA speciescontaining the bcl-6 sequence; and (d) comparing the RNA speciesobtained from (c) with the RNA species from a known normal subject, thedifference in size of the species indicating the occurrence of B-celllymphoma in the subject.

In an embodiment, the size fractionation in step (b) is effected by apolyacrylamide or agarose gel.

This invention also provides the above-described method where in step(c), the nucleic acid molecule is labeled with a detectable marker. Thedetectable marker includes but is not limited to a radiolabelledmolecule, a fluorescent molecule, an enzyme, or a ligand.

This invention also provides the above-method further comprisestransferring the RNA species into a solid matrix before step (c).

This invention also provides various uses of bcl-6 locus/gene an itsderivatives. This invention further provides a method for diagnosis of Bcell lymphoma and/or diffuse-type B cell lymphoma using bcl-6 DNA probesor synthetic oligonucleotide primers derived from bcl-6 sequences todetect bcl-6 rearrangements/mutations by Southern blotting PCR or otherDNA based techniques.

This invention also provides a method of diagnosis of B cell lymphomaand/or diffuse-type B cell lymphoma using bcl-6 DNA probes or syntheticoligonucleotide primers derived from bcl-6 sequences to detect abnormalbcl-6 RNA species by Northern blotting, PCR or other RNA-basedtechniques.

This invention further provides a method of diagnosis of B cell lymphomaand/or diffuse-type B cell lymphoma using antiserum or monoclonalantibodies directed against the bcl-6 protein product(s).

This invention provides a method of treating a subject withnon-Hodgkin's lymphoma comprising administering an effective amount ofthe antisense molecule of the nucleic acid molecule bcl-6 operativelylinked to a suitable regulatory element coupled with a therapeutic DNAinto a tumor cell of a subject, thereby treating the subject withnon-Hodgkin's lymphoma.

This invention provides a method of treating a subject withnon-Hodgkin's lymphoma, comprising administering an effective amount ofthe antagonist capable of blocking the expression of the polypeptideencoded by the isolated nucleic acid molecule of bcl-6, and a suitableacceptable carrier, thereby treating the subject with non-Hodgkin'slymphoma.

Further, as is known to those of ordinary skill in the art effectiveamounts vary with the type of therapeutic agent. It is known to those ofordinary skill in the art how to determine an effective amount of asuitable therapeutic agent.

The preparation of therapeutic compositions which contain polypeptides,analogs or active fragments as active ingredients is well understood inthe art. Typically, such compositions are prepared as injectables,either as liquid solutions or suspensions, however, solid forms suitablefor solution in, or suspension in, liquid prior to injection can also beprepared. The-preparation can also be emulsified. The active therapeuticingredient is often mixed with excipients which are pharmaceuticallyacceptable and compatible with the active ingredient. Suitableexcipients are, for example, water, saline, dextrose, glycerol, ethanol,or the like and combinations thereof. In addition, if desired, thecomposition can contain minor amounts of auxiliary substances such aswetting or emulsifying agents, pH buffering agents which enhance theeffectiveness of the active ingredient.

A polypeptide, analog or active fragment can be formulated into thetherapeutic composition as neutralized pharmaceutically acceptable saltforms. Pharmaceutically acceptable salts include the acid addition salts(formed with the free amino groups of the polypeptide or antibodymolecule) and which are formed with inorganic acids such as, forexample, hydrochloric or phosphoric acids, or such organic acids asacetic, oxalic, tartaric, mandelic, and the like. Salts formed from thefree carboxyl groups can also be derived from inorganic bases such as,for example, sodium, potassium, ammonium, calcium, or ferric hydroxides,and such organic bases as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine, and the like.

The subjects contained herein may be a mammal, or more specifically ahuman, horse, pig, rabbit, dog, monkey, or rodent. In the preferredembodiment the subject is a human.

The compositions are administered in a manner compatible with the dosageformulation, and in a therapeutically effective amount. Precise amountsof active ingredient required to be administered depend on the judgmentof the practitioner and are peculiar to each individual.

Suitable regimes for initial administration and booster shots are alsovariable, but are typified by an initial administration followed byrepeated doses at one or more hour intervals by a subsequent injectionor other administration.

As used herein administration means a method of administering to asubject. Such methods are well known to those skilled in the art andinclude, but are not limited to, administration topically, parenterally,orally, intravenously, intramuscularly, subcutaneously or by aerosol.Administration of the agent may be effected continuously orintermittently such that the therapeutic agent in the patient iseffective to treat a subject with non-hodgkin's lymphoma.

Finally, this invention provides a therapy of B cell lymphoma and/ordiffuse-type B cell lymphoma using anti bcl-6 reagents includingspecific antisense sequences and compounds interfering with bcl-6functions.

This invention will be better understood from the Experimental Detailswhich follow. However, one skilled in the art will readily appreciatethat the specific methods and results discussed are merely illustrativeof the invention as described more fully in the claims which followthereafter.

EXPERIMENTAL DETAIL SECTION I

Materials and Methods

DNA Extraction and Southern Blot Analysis. Total genomic DNA waspurified from frozen tumor biopsies by cell lysis, proteinase Kdigestion, "salting-out" purification and ethanol precipitation aspreviously described (11). Southern blot hybridization analysis wasperformed in 50% formamide, 3× SSC, 10× dextran sulphate, 5× Denhardt'ssolution, 0.5% SDS at 37° C. for 16 hrs. Filters were washed in 0.2×SSC, 0.5% SDS at 60° C. for 2 hrs. DNA probes were ³² P-labelled by therandom priming method (12).

DNA Probes. The following probes were used for Southern blot analysis ofIg gene rearrangements: i) (J_(H)) probe: 6.6 kb BamHi/HindIII fragmentfrom the human Ig heavy-chain (Ig_(H)) locus (13); ii) (C.sub.μ) probe:1.3 kb EcoRI fragment containing the first two exons of human C.sub.μ(13).

Genomic Cloning. Genomic libraries from NHL cases SM1444 and KC1445 wereconstructed by partial Sau 3A restriction digestion of genomic DNA andligation of gel-purified 15-20 kb fractions into LambdaGem-11 phagevector (Promega). Library screening was performed byplaque-hybridization using the C.sub.μ probe.

Fluorescence in situ Hybridization Analysis (FISH). Phage DNA waslabelled with biotin-14-dATP by nick translation and hybridized tometaphase spreads from normal human lymphocytes as described (14). Tovisualize the hybridization signal and the corresponding bandssequentially under the microscope, the slides were stained andcounterstained with propidium iodide and 4'6'-diamideno-2-phenylindole(DAPI), respectively.

Northern Blot Hybridization Analysis. RNAs from several human cell lineswere extracted by the guanidine-isothiocyanate method (15). For Northernblot analysis, RNA samples were electrophoresed through 0.9%agarose-2.2M formaldehyde gels and then transferred to nitrocellulosefilters. Hybridization and washing were performed as described forSouthern blot analysis.

Experimental Results

DNA was extracted from tumor tissue of two cases (SM1444 and KC1445) ofIgM-producing, diffuse-type B-cell NHL carrying the t(3;14)(q27;q32)translocation. Since the involvement of the Ig_(H) locus was suspectedbased on the 14q32 breakpoint, SM1444 and KC1445 DNAs were firstanalyzed by Southern blot hybridization using combinations of enzymesand probes specific for the J_(H) and C.sub.μ regions of the Ig_(H)locus (13). In both cases, digestion by BamHI showed rearrangedfragments containing J_(H) sequences (FIG. 1). Subsequent hybridizationsto the C.sub.μ probe showed, in each case, that one rearranged fragmentcontaining J_(H) sequences was not linked to C.sub.μ sequences (seefailure of the C.sub.μ probe to hybridize to the same rearranged BamHIfragment detected by J_(H) (FIG. 1) as would be expected for thephysiologically rearranged Ig_(H) allele in IgM producing cells. Inaddition, in both cases, digestion with HindIII and hybridization withC.sub.μ detected a rearranged fragment, a finding inconsistent witheither germ-line or physiologically rearranged Ig_(H) genes, since bothHindIII sites flanking C.sub.μ sequences are not involved in V-D-Jarrangements (13). The observed pattern is, however, consistent withchromosomal breakpoints located within C.sub.μ switch sequences, aspreviously observed in several cases of chromosomal translocationsinvolving the Ig_(H) locus (2,16-18).

Based on this analysis, the C.sub.μ containing fragments from each casewere cloned by screening genomic libraries constructed from SM1444 andKC1445 DNAs using the C.sub.μ probe. Restriction mapping andhybridization analysis of several phage clones led to the identificationof recombinant phages from each library which contained C.sub.μsequences linked to sequences unrelated to the Ig_(H) locus (see FIG. 2for maps of representative phage clones). The Ig portions of the phageinserts overlapped along the C.sub.μ region extending 5' into the switchregion where alignment with the restriction map of the normal Igheavy-chain locus was lost. The location of the breakpoint withinC.sub.μ switch sequences was confirmed for case SM1444 by DNA sequenceanalysis of the breakpoint junction of phage SM-71, which revealed thepresence of the repeated motifs typical of the Ig_(H) switch regions onthe chromosome 14 side (19). The Ig-unrelated portions of phage SM-71and KC-51 also overlapped with each other in their restriction maps,suggesting that they were derived from the same genomic region. Thisnotion is further supported by the fact that probe Sac 4.0 derived fromSM-71 was able to hybridize to the corresponding region of KC-51 inSouthern blot analysis.

To determine the chromosomal origin of the Ig-unrelated sequences, arecombinant phage (SM-71) derived from case SM1444, was used as a probein FISH analysis on metaphase chromosome spreads from mitogen-stimulatednormal blood lymphocytes. The phage probe hybridized specifically tochromosome 14q32 as well as to chromosome 3q27 (FIGS. 3A-3B), indicatingthat the recombinant phage insert contained one of the two chromosomaljunctions of the reciprocal t(3;14) translocation. Thus, taken together,the results of cloning and FISH analysis established that, in both NHLcases studied, the chromosomal translocation has linked sequences withinthe switch region of the C.sub.μ locus to sequences from band 3q27,consistent with the cytogenetic description of the t(3;14) (q27;q32)translocation. In the two NHL cases studied, the breakpoints on 3q27were located within 3 kb of the same genomic locus, which was termedbcl-6.

In order to determine whether 3q27 breakpoints in additional NHL caseswere also located within the cloned portion of the bcl-6 locus, bcl-6rearrangements were examined in a total of 19 NHL cases carrying 3q27breakpoints, including 4 (two cloned cases and two additional ones)carrying t(3;14) (q27;q32) as well as 15 cases carrying 3q27translocations involving regions other than 14q32. Southern blothybridization using probes derived from phage SM-71 (see FIG. 2)detected rearranged fragments in EcoRI-and/or BglII-digested DNA in 7 of19 cases studied, including all 4 t(3;14) cases as well as 3 cases withother types of translocations (see FIGS. 4A-4C for cytogeneticdescription of the cases and representative results). These resultsindicate that heterogeneous 3q27 breakpoints cluster in a fairlyrestricted region within bcl-6 independently of the partner chromosomeinvolved in the translocation.

Whether the bcl-6 locus adjacent to the chromosomal breakpointscontained a transcriptional unit was investigated. Probe Sac 4.0 (seeFIG. 2) was used to detect RNA expression in several human cell lines byNorthern blot analysis. A major 2.4 kb RNA species was readilydetectable in two B-cell derived cell lines tested, while a relativelyless abundant 4.4 kb species is present in CB33 only. No hybridizationwas detected in a T-cell derived cell line (HUT 78) nor in HeLa cells(FIG. 5). This result indicates that 3q27 sequences immediately adjacentto the chromosomal breakpoint cluster are part of a gene (bcl-6) whichis expressed in cells of the B lineage.

Experimental Discussion

This study reports the identification and cloning of a genomic region,bcl-6, involved in recurrent chromosomal translocations affecting band3q27 in NHL. The region is defined by the clustered position ofbreakpoints in seven NHL cases carrying 3q27 translocations involvingeither IgH or several other loci. A more precise definition of the bcl-6locus and of the frequency of its involvement in NHL requires cloningand characterization of additional bcl-6 sequences and studyingadditional tumor cases. Nevertheless, the finding that varioustranslocation partner chromosomes have been joined to the same region onchromosome 3 in cytogenetically heterogenous NHL cases supports thenotion that rearrangement of the bcl-6 locus may represent the criticalcommon denominator of translocations involving 3q27.

The second finding of this study is that the bcl-6 locus contains a genewhich is expressed in B-cells. It is not clear at this stage whether thechromosomal breakpoints directly truncate coding or regulatory sequencesof bcl-6, or, whether the gene remains intact with its regulationoverridden by transcriptional control motifs juxtaposed by thetranslocation. The clustering of breakpoints in the seven studied NHLcases suggests, however, that bcl-6 may be a proto-oncogene which cancontribute to NHL pathogenesis upon activation by chromosomaltranslocation. Results of this study will allow elucidation of thenormal structure and function of the bcl-6 gene in order to understandthe pathogen consequences of chromosomal translocation of bcl-6 and itsrole in lymphomagenesis.

REFERENCES FOR SECTION I

1. Gaidano, G., and Dalla-Favera, R., (1992) Oncogenes and tumorsuppressor genes. In: Neoplastic Hematopatholovy, D. M. Knowles (ed.),Wilkins & Wilkins, pp 245-261.

2. Dalla-Favera, R., et al. (1982) Human c-myc oncogene is located onthe region of chromosome 8 that is translocated in Burkitt lymphomacells, Proc. Natl. Acad. Sci. USA 79:7824-7827.

3. Taub, R., et al. (1982) Translocation of c-myc gene into theimmunoglobulin heavy chain locus in human Burkitt lymphoma and murineplasmacytoma cells, Proc. Natl. Acad. Sci. USA 79:7837-7841.

4. Bakhshi, A., et al. (1985) Cloning the chromosomal breakpoint oft(14;18) human lymphomas: clustering around J_(H) on chromosome 14 andnear a transcriptional unit on 18, Cell 41:889-906.

5. Tsujimoto, U., et al. (1985) Involvement of the Bcl-2 gene in humanfollicular lymphoma, Science 228:1440-1443.

6. Cleary, M. L., and Sklar, J., (1985) Nucleotide sequence of at(14;18) chromosomal breakpoint in follicular lymphoma and demonstrationof a breakpoint-cluster region near a transcriptionally active locus onchromosome 18, Proc. Natl. Acad. Sci. USA 82:7439-7444.

7. Motokura, T., et al. (1991) A novel cyclin encoded by a bcl-1 linkedcandidate oncogene, Nature 350:512-514.

8. Raffeld, M., and Jaffe, E. S., (1991) Bcl-1, t(11;14), and mantlezone lymphomas, Blood 78:259-261.

9. Offit, K., et al. (1989) t(3;22) (q27;q11): A novel translocationassociated with diffuse non-Hodgkin's lymphoma, Blood 74:1876-1879.

10. Bastard, C., et al. (1992) Translocations involving band 3q37 and Iggene regions in non-Hodgkin's lymphoma, Blood 79:2527-2531.

11. Miller, S. A., et al. (1988) A simple salting out procedure forextracting DNA from human nucleated cells, Nucleic Acids Res,16:1215-1218.

12. Feinberg, A. P., and Vogelstein, B., (1983) A technique forradiolabelling DNA restriction endonuclease fragments to high specificactivity, Anal. Biochem., 132:6-13.

13. Ravetch, J. V., et al. (1981) Structure of the human immunoglobulinμ locus: characterization of embryonic and rearranged J and D regions,Cell, 27:583-591.

14. Rao, P. H., et al. (1994) Subregional localization of 20 single-copyloci to chromosome 6 by fluorescence in situ hybridization, Cyto. andCell Genetics 66:272-273.

15. Chirgwin, J. M., et al. (1979) Isolation of biologically activeribonucleic acid from sources enriched in ribonuclease, Biochemistry,18:5294-5299.

16. Peschle, C., et al. (1984) Translocation and rearrangement of c-mycinto immunoglobulin alpha heavy chain locus in primary cells from acutelymphocytic leukemia, Proc. Natl. Acad. Sci. U.S.A., 81:5514-5518.

17. Showe, L. C., et al. (1985) Cloning and sequencing of a c-myconcogene in a Burkitt's lymphoma cell line that is translocated to agerm line alpha switch region, Mol. Cell. Biol., 5:501-509.

18. Neri, A., Barriga, et al. (1988) Different regions of theimmunoglobulin heavy chain locus are involved in chromosomaltranslocations in distinct pathogenic forms of Burkitt lymphoma, Proc.Natl. Acad. Sci. USA, 85:2748-2752.

19. Rabbits, T. H., et al. (1991) Human immunoglobulin heavy chaingenes: evolutionary comparisons of C mu, C delta and C gamma genes andassociated switch sequences, Nucleic Acids Res., 9:4509-4524.

20. Schmid, et al. (1991) Nature, 332:733.

EXPERIMENTAL DETAIL SECTION II

Introduction

The molecular analysis of specific chromosomal translocations hasimproved the understanding of the pathogenesis of non-Hodgkin lymphoma(NHL), a heterogeneous group of B-cell or, less frequently, T-cellmalignancies (1,2). The (14;18) chromosomal translocation, which causesthe deregulated expression of the anti-apoptosis gene BCL-2, plays acritical role in the development of follicular lymphoma (FL) (3-6),which accounts for 20 to 30% of all NHL diagnoses (7). Burkitt'slymphoma (BL) and mantle-cell lymphoma, two relatively rare NHL types,are characterized by chromosomal translocations causing the deregulatedexpression of the cell-cycle progression genes C-MYC and theBCL-1/cyclin D1, respectively (8-15).

Relatively little is known about the molecular pathogenesis of diffuselarge cell lymphoma (DLCL), the most frequent and most lethal humanlymphoma (7). DLCL accounts for .sup.˜ 40% of initial NHL diagnoses andis often the final stage of progression of FL(7). A small percentage ofDLCL display C-MYC rearrangements (16) and 20 to 30% display alterationsof BCL-2 reflecting the tumor's derivation from FL (17). However, noconsistent molecular alteration has been identified that is specific forDLCL.

Chromosomal translocations involving reciprocal recombinations betweenband 3q27 and several other chromosomal sites are found in 8 to 12% ofNHL cases, particularly in DLCL (18-19). From NHL samples displayingrecombinations between 3q27 and the immunoglobulin (Ig) heavy chainlocus on 14q32, the chromosomal junctions of several (3;14)(q27;q32)translocations were cloned and identified a cluster of breakpoints at a3q27 locus named BCL-6.

Experimental Results

To isolate normal BCL-6 cDNA, a cDNA library constructed form the NHLcell line Bjab (22) was screened with a probe (20-21) derived from thechromosomal region flanking the breakpoints of two t(3;14)(q27;32)cases. A phage cDNA library constructed from RNA of the Bjab lymphomacell line was screened (1×10⁶ plaques) by plaque hybridization with theSac 4.0 probe that had been ³² P-labelled by random priming (22).Sequence analysis (FIGS. 10A-10B) revealed that the longest clone (3549bp), approximately the same size as BCL-6 RNA, codes for a protein of706 amino acids with a predicted molecular mass of 79kD. The putativeATG initiation codon at position 328 is surrounded by a Kozak consensussequence (23) and is preceded by three upstream inframe stop codons. The1101-bp 3'-untranslated region contains a polyadenylation signalfollowed by a track of poly(A). These features are consistent with BCL-6being a functional gene.

The NH₂ -- and COOH-- termini of the BCL-6 protein (FIGS. 10A-10B) havehomologies with "zinc-finger" transcription factors (24). BCL-6 containssix C₂ H₂ zinc-finger motifs (FIG. 10A) and a conserved stretch of sixamino acids (the H/C link) connecting the successive zinc-finger repeats(25), BCL-6 can be assigned to the Kruppel-like subfamily of zinc-fingerproteins. The NH₂ -- terminal region of BCL-6 is devoid of the FAX (27)and KRAB (28) domains sometimes seen in Kruppel-related zinc-fingerproteins, but it does have homologies (FIG. 11) with other zinc-fingertranscription factors including the human ZFPJS protein, a putativehuman transcription factor that regulates the major histocompatibilitycomplex II promoter, the Tramtrack (ttk) and Broad-complex (Br-c)proteins in Drosophila that regulate developmental transcription (29),the human KUP protein (31), and the human PLZF protein, which isoccasionally involved in chromosomal translocations in humanpromyelocytic leukemia (32). The regions of NH₂ -terminal homology amongZFPJS, ttk, Br-c, PLZF and BCL-6 also share some degree of homology withviral proteins (e.g. VA55R) of the poxvirus family (33) as well as withthe Drosophila kelch protein involved in nurse cell-oocyte interaction(34). These structural homologies suggest that BCL-6 may function as aDNA-binding transcription factor that regulates organ development andtissue differentiation.

The cDNA clone was used as a probe to investigate BCL-6 RNA expressionin a variety of human cell lines by Northern blot analysis. A single 3.8kb RNA species was readily detected in cell lines derived from matureB-cells, but not from pro-B-cells or plasma cells, T cells or otherhematopoietic cell lineages. The BCL-6 RNA was not detectable in othernormal other tissues, except for skeletal muscle in which low levelexpression was seen. Thus, the expression of BCL-6 was detected inB-cells at a differentiation stage corresponding to that of DLCL cells.This selective expression in a "window" of B-cell differentiationsuggests that BCL-6 plays a role in the control of normal B-celldifferentiation and lymphoid organ development.

To characterize the BCL-6 genomic locus, the same cDNA probe to screen agenomic library from human placenta was used. A phage genomic libraryconstructed from normal human placenta DNA (Stratagene) was screened(8×10⁵ plaques) with the BCL-6 cDNA. Twelve overlapping clones spanning.sup.˜ 50kb of genomic DNA were isolated. After restriction mapping, theposition of various BCL-6 exons was determined by Southern hybridizationusing various cDNA probes. By restriction mapping, hybridization withvarious CDNA probes, and limited nucleotide sequencing, the BCL-6 genewas found to contain at least ten exons spanning .sup.˜ 26 kb of DNA(FIG. 12). Sequence analysis of the first and second exons indicatedthat they are noncoding and that the translation initiation codon iswithin the third exon.

Various cDNA and genomic probes were used in Southern (DNA) blothybridizations to determine the relationship between 3q27 (Table 1).Monoallelic rearrangements of BCL-6 were detected in 12 of 17 tumors byusing combinations of restriction enzymes (Bam HI and Xba I) and probewhich explore .sup.˜ 16 kb within the BCL-6 locus. These 12 positivecases carry recombinations between 3q27 and several differentchromosomes (Table 1), indicating that heterogeneous 3q27 breakpointscluster in a restricted genomic locus irrespective of the partnerchromosome involved in the translocation. Some DLCL samples (5 of 17) donot display BCL-6 rearrangements despite cytogenetic alterations in band3q27, suggesting that another gene is involved or, more likely, thatthere are other breakpoint clusters 5' or 3' to BCL-6. If the latter istrue, the observed frequency of BCL-6 involvement in DLCL (33%, seebelow) may be an underestimate.

                  TABLE 1    ______________________________________    Frequency of BCL-6 rearrangements in DLCL carrying    chromosomal translocations affecting band 3q27                       Fraction of tumors with BCL-6    Translocation      rearrangements    ______________________________________    t (3;14 (q27;q32)  4/4    t (3;22) (q27;q11) 2/3    t (3;12) (q27;q11) 1/1    t (3;11) (q27;q13) 1/1    t (3;9) (q27;p13)  0/1    t (3;12) (q27;q24) 0/1    der (3) t (3;5) (q27;q31)                       1/1    t (1;3) (q21;q27)  1/1    t (2;3) (q23;q27)  1/1    der (3) t (3;?) (q27;?)                       1/3    ______________________________________

Tumor samples listed in the Table were collected and analyzed forhistopathology and cytogenetics at Memorial Sloan-Kettering CancerCenter.

A panel of tumors not previously selected on the basis of 3q27breakpoints but representative of the major subtypes of NHL as well asof other lymphoproliferative diseases was analyzed. Similarrearrangements were detected in 13 of 39 DLCL, but not in other casesincluding other NHL subtypes (28 FL, 20 BL, and 8 small lymphocyticNHL), acute lymphoblastic leukemia (ALL; 21 cases), and chroniclymphocytic leukemia (CLL; 31). These findings indicate that BCL-6rearrangements are specific for and frequent in DLCL. In addition, thefrequency of rearrangements in DLCL (33%) significantly exceeds that (8to 12%) reported at the cytogenetic level, suggesting that some of theobserved rearrangements may involve submicroscopic chromosomalalterations undetectable at the cytogenetic level.

All the breakpoints in BCL-6 mapped to the putative 5' flanking region,the first exon or the first intron (FIG. 12). For two patients thatcarry (3;12)(q27;q32) translocations, the chromosomal breakpoints havebeen cloned and precisely mapped to the first intron (SM1444) or to 5'flanking sequences (KC1445) of BCL-6 on 3q27, and to the switch regionof IgH on 14q32 (20-21). In all rearrangements, the coding region ofBCL-6 was left intact whereas the 5' regulatory region, presumablycontaining the promoter sequences, was either completely removed ortruncated. The resultant fusion of BCL-6 coding sequences toheterologous (from other chromosomes) or alternative (within the BCl-6locus) regulatory sequences may disrupt the gene's normal expressionpattern. A BCL-6 transcript of normal size was detected by Northern blotanalysis of DLCL cells carrying either normal or truncated BCL-6. Someof the truncations were in the 5' flanking sequences and would thereforenot be expected to generate structurally abnormal transcripts.

Experimental Discussion

Zinc-finger encoding genes are candidate oncogenes as they have beenshown to participate in the control of cell proliferation,differentiation, and organ pattern formation (24). In fact, alterationsof zinc-finger genes have been detected in a variety of tumor types.These genes include PLZF (32) and PML (35-38) in acute promyelocticleukemia, EVI-1 (38-39) in mouse and human myeloid leukemia, TTG-1 (40)in T-cell CLL, HTRX (41-43) in acute mixed-lineage leukemia, and WT-1(44) in Wilm's tumor. Terminal differentiation of hematopoietic cells isassociated with the down-regulation of many Kruppel-type zinc-fingergenes. Thus, constitutive expression of BCL-6, caused by chromosomalrearrangements, interferes with normal B-cell differentiation, therebycontributing to the abnormal lymph node architecture typifying DLCL.

Given that DLCL accounts for .sup.˜ 80% of NHL mortality (7), theidentification of a specific pathogenetic lesion has importantclinicopathologic implications. Lesions in BCL-6 may help in identifyingprognostically distinct subgroups of DLCL. In addition, since atherapeutic response can now be obtained in a substantial fraction ofcases (7), a genetic marker specific for the malignant clone may be acritical tool for the monitoring of minimal residual disease and earlydiagnosis of relapse (45).

The gene cloned from chromosomal translocations affecting band 3q27,which are common in DLCL codes for a 79 kD protein that is homologouswith zinc-finger transcription factors. In 33% (13/39) of DLCL samples,but not in other types of lymphoid malignancies, the BCL-6 gene istruncated within its 5' noncoding sequences, suggesting that itsexpression is deregulated. Thus, BCL-6 is a proto-oncogene specificallyinvolved in the pathogenesis of DLCL.

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39. Fichelson, S., et al. (1992) Leukemia 6:93.

40. McGuire, E. A., et al. (1989) Cell Biol. 9:2124.

41. Djabali, M., et al. (1992) Nature Genet. 2:113.

42. Tkachuk, D. C., et al. (1992) Cell 71:691.

43. Gu, Y., et al. (1992) Cell 71:701.

44. Haber, D. A., et al. (1990) Cell 61:1257.

45. Medeiros, L. J., et al. (1992) Neoplastic Hemopathology, pp.263-298.

EXPERIMENTAL DETAIL SECTION III

Introduction

Non Hodgkin's lymphoma (NHL), the most frequent tumor occurring inpatients between the ages of 20 and 40, includes several distinctclinico-pathologic subtypes, among which diffuse lymphoma with a largecell component (DLLC) is the most clinically relevant in terms ofmorbidity and mortality (1). DLLC include intermediate-grade lymphomaswith pure diffuse large-(DLCL), or mixed small- and large-cell (MX-D)histology, as well as high-grade immunoblastic (IMB) lymphoma. Thesetumors can occur "de novo ", accounting for 30-40% of initial NHLdiagnosis and, in addition, can represent the final "transformation"stage of follicular lymphomas (FL), small lymphocytic lymphoma andchronic lymphocytic leukemia. Considered together, "de novol" and"post-transformation" DLLC account for up to 80% of NHL mortality (1).

During the past decade, abnormalities involving proto-oncogenes andtumor suppressor genes have been identified in association with distinctNHL subtypes (2). These genetic lesions represent important steps inlymphomagenesis as well as tumor-specific markers which have beenexploited for diagnostic and prognostic purposes (3,4). Examples includealterations of the MYC oncogene in Burkitt lymphoma (BL), and of theBCL-2 and BCL-1 oncogenes in FL and mantle-cell NHL, respectively. Withrespect to DLLC, several molecular alterations have been detected atvariable frequency, but none has been specifically or consistentlyassociated with the disease (2). In this invention the frequency anddisease-specificity of BCL-6 (5-10) rearrangements among the principalcategories of lymphoproliferative disease, including different NHLsubtypes, acute and chronic lymphoid leukemias and multiple myeloma isdemonstrated.

Materials and Methods

Samples of lymphnode biopsies, bone marrow aspirates and peripheralblood were collected by standard diagnostic procedures during the courseof routine clinical evaluation in the Division of Surgical Pathology,Department of Pathology, Columbia University. In all instances, thespecimens were collected before specific anti-tumor treatment. Diagnoseswere based on the results of histopathologic, immunophenotypic andimmunogenotypic analysis (11). In all cases, the fraction of malignantcells in the pathologic specimen was at least 70% as determined bycytofluorimetric or immunohistochemical analysis of cell-surface markersor antigen receptor (immunoglobulin heavy chain and T cell receptor βchain) gene rearrangement analysis (11).

Genomic DNA was prepared from diagnostic specimens by cell lysis,proteinase K digestion, phenol-chloroform extraction and ethanolprecipitation. For Southern blot analysis, 6 μg of DNA were digestedwith the appropriate restriction endonuclease, electrophoresed in a 0.8%agarose gel, denatured, neutralized and transferred to Duralose filters(Stratagene, La Jolla, Calif.). Filters were then hybridized with theBCL-6-specific Sac 4.0 probe (10) that had been ³² P-labelled by therandom priming technique. After hybridization, filters were washed in0.2× SSC (1× SSC=0.15M NaCl+0.015M sodium citrate / 0.5% sodium dodecylsulfate) for 2 hours at 60° C. and then subjected to autoradiography for24-48 hours at -80° C. using intensifying screens.

All NHL cases were also analyzed for rearrangement of the BCL-2 geneusing the previously described probes corresponding to the MBR and MCRregions. Immunophenotypic analysis of immunoglobulin and cell surfacemarker expression was performed as previously described (11).

Comparisons of histologic subsets with or without BCL-6 rearrangementwere made utilizing the method of inferences from proportions (12).

Experimental Results

The tumor panel (Table 2) used for this study is representative of themajor categories of lymphoproliferative disease including NHL, 125cases, ALL 45, CLL 51 and MM 23. The NHL series was representative oflow- 41, intermediate- 45 and high-grade 24 subtypes according to theWorking Formulation. Fifteen cases of cutaneous T-cell NHL were alsoincluded.

The presence of BCL-6 rearrangements was analyzed by Southern blothybridization of tumor DNAs using a probe (Sac 4.0) (10) and restrictionenzymes (BamHI and XbaI) which, in combination, explore a region of 15.2Kb containing the 5' portion of the BCL-6 gene (first exon, 7.5 Kb offirst intron and 7.4 Kb of 5' flanking sequences) (10). This region waspreviously shown to contain the cluster of breakpoints detected in NHL.No additional rearrangements were found using probes and restrictionenzymes exploring approximately 10 kb either 5' or 3' to BCL-6 sequences

The results of this analysis are summarized in Table 2 andrepresentatively shown in FIGS. 13A-13B. All cases of ALL, CLL and MMshowed a normal BCL-6 gene.

Eighteen of the 125 NHL cases displayed BCL-6 rearrangements. Amongdistinct NHL histologic subtypes, rearrangements were detected in 16/45(35.5%) DLLC and in 2/31 (6.4%) FL (p<0.001). One of these 2 FL casesshowed both follicular and diffuse patterns of growth. Among DLLC,rearrangements were significantly more frequent in DLCL (15/33, 45.4%)than in MX-D (1/10, 10%) (p<0.01), suggesting that these genetic lesionsmay be specifically associated with the diffuse large cell component ofthese tumors. All of the DLLC cases displaying BCL-6 rearrangementslacked BCL-2 rearrangements which were found in only two 2 DLLC cases.Although cytogenetic data were not available for the panel of tumorsstudied, the frequency of BCL-6 rearrangements far exceeds that expectedfor 3q27 aberrations (10-12% in DLLC) (8, 9), suggesting that BCL-6rearrangements can occur as a consequence of submicroscopic chromosomalaberrations.

In order to determine whether the presence of BCL-6 rearrangementscorrelated with distinct immunophenotypic features of DLLC, the entirepanel was analyzed for expression of immunoglobulin κ and λ lightchains, and B cell-associated antigens CD19, CD20 and CD22 (11). Asexpected, the expression of these markers was variable in the DLLC casestested. However, no correlation with the BCL-6 rearrangement status wasfound.

                  TABLE 2    ______________________________________    Rearrangements of the BCL-6 gene in lymphoid tumors    TUMOR      HISTOTYPE  REARRANGED/TESTED                                          %    ______________________________________    NHL    Low grade: SL         0/10            0               SCC-F      2*/18           11               MX-F       0/13            0    Intermediate grade:               MX-D       1/10            10               DLCL       15/33           45               SCC-D      0/2             0    High grade:               IMB        0/2             0               SNCL       0/22            0    Others:    CTCL       0/15            0    ALL        B-lineage: 0/34            0               T-lineage: 0/11            0    CLL        B-lineage: 0/41            0               T-lineage: 0/10            0    MM                    0/23            0    ______________________________________     NHL, nonHodgkin's lymphoma; ALL, acute lymphoblastic leukemia; CLL,     chronic lymphocytic leukemia; MM, multiple myeloma; SL, small lymphocytic     SCCF, follicular small cleaved cell; MXF, follicular mixed; MXD, diffuse     mixed cell; DLCL, diffuse large cell; SCCD, diffuse small cleaved cell;     IMB, immunoblastic; SNCL, small noncleaved cell lymphoma; CTCL, cutaneous     Tcell lymphoma. *: one case showed follicular and diffuse growth patterns

Experimental Discussion

In this study, BCL-6 rearrangement is established as the most frequentabnormality detectable in DLLC. Previous studies have indicated MYC andBCL-2 rearrangements detectable in 5-20% and 20% of DLLC, respectively(13). Compared to those lesions, which are also commonly associated withBurkitt's lymphoma (MYC) and FL (BCL-2), BCL-6 rearrangements appear tobe more disease-specific since they were exclusively found in DLLC withthe exception of 2 of 45 FL cases. Considering that one of these two FLcases displayed areas of diffuse histology, it is conceivable that BCL-6rearrangements may be occasionally associated with atypical FL caseswith mixed follicular and diffuse components. The recurrent and specificassociation between DLLC and structural lesions of a gene coding for azinc finger-type transcription factor related to several knownproto-oncogenes 10 suggests that these abnormalities may play a role inpathogenesis of DLCL.

Among the heterogeneous DLLC spectrum, BCL-6 rearrangements weresignificantly more frequent in tumors displaying a pure diffuse largecell histology (DLCL) all of which lacked BCL-2 rearrangements.Considering that DLCL can originate both "de novo" and from the"transformation" of FL, and that the latter typically carry BCL-2rearrangements, results suggest that BCL-6 rearrangements may bespecifically involved in the pathogenesis of "de novol" DLLC. Thisconclusion is consistent with recent findings indicating that othergenetic alterations, namely the inactivation of the p53 tumor suppressorgene, may be involved in the transformation of FL to DLLC (14).

The results presented herein have relevant diagnostic and prognosticimplications. DLLC represent a heterogeneous group of neoplasms whichare treated homogeneously despite the fact that only 50% of patientsexperience long-term disease free survival (1). The presence of a markersuch as BCL-6 rearrangement identifies a sizable subset of cases with adistinct pathogenesis and, distinct biological behavior.

The pathogenesis of non-Hodgkin lymphoma with a large-cell component(DLLC, including diffuse large-cell, DLCL, diffuse mixed-cell, MX-D, andimmunoblastic, IMB) is unknown. The incidence and disease-specificity ofBCL-6 rearrangements in a large panel of lymphoid tumors, includingacute and chronic lymphoid leukemias (96 cases), various NHL types (125cases), and multiple myelomas (23 cases) has been tested. BCL-6rearrangements were found in 16/45 (35.5%) DLLC, more frequently in DLCL(15/33, 45%) than in MX-D (1/10, 10%), in 2/31 (6.4%) follicular NHL,and in no other tumor types. BCL-6 rearrangements represent the firstgenetic lesion specifically and recurrently associated with DLLC andshould prove useful for understanding the pathogenesis as well as forthe clinical monitoring of these tumors.

REFERENCES FOR SECTION III

1. Magrath, I. T. (1990) The Non-Hodgkin's Lymphomas:

An Introduction, The Non-Hodgkin's Lymphomas, Edward Arnold, London, p1.

2. Gaidano, G., and Dalla-Favera, R. (1993) Biologic and molecularcharacterization of non-Hodgkin's lymphoma, Curr. Opin. Oncol. 5:776.

3. Gribben, J. G., et al. (1991) Immunologic purging of marrow assessedby PCR before autologous bone marrow transplantation for B-celllymphoma, N. Engl. J. Med. 325:1525.

4. Yunis, J. J., et al. (1989) Bcl-2 and other genomic alterations inthe prognosis of large-cell lymphoma, N. Enql. J. Med. 320:1047.

5. Ye, B. H., et al. (1993) Cloning of bcl-6, the locus involved inchromosome translocations affecting band 3q27 in B-cell lymphoma, CancerRes. 53:2732.

6. Baron, B. W., et al. (1993) Identification of the gene associatedwith the recurring chromosomal translocations t(3;14)(q27;q32) andt(3;22) (q27;q11) in B-cell lymphomas, Proc. Natl. Acad. Sci. USA 90:5262.

7. Kerckaert, J. P., et al. (1993) LAZ3, a novel zinc-finger encodinggene, is disrupted by recurring chromosome 3q27 translocations in humanlymphomas, Nature Genet. 5:66.

8. Offit, K, et al. (1989) t(3;22 (q27;q11): A novel translocationassociated with diffuse non-Hodgkin's lymphoma, Blood 74: 1876.

9. Bastard, C, et al. (1992) Translocations involving band 3q27 and Iggene regions in non-Hodgkin's lymphoma, Blood 79: 2527.

10. Ye, B. H., et al. (1993) Alterations of a zinc-finger encoding gene,BCL-6, in diffuse large-cell lymphoma, Science 262:747.

11. Knowles, D. M. (ed.) (1992) Neoplastic Hemopathology, Williams &Wilkins, Baltimore, Md.

12. Armitage, P., (1977) Statistical methods in medical research,Blackwell Scientific Publications, London, p. 11.

13. Chaganti, R. S. K., et al. (1989) Specific translocations innon-Hodgkin's lymphoma: incidence, molecular detection, and histologicaland clinical correlations, Cancer Cells 7:33.

14. Lo Coco, F., et al. (1993) p53 mutations are associated withhistologic transformation of follicular lymphoma, Blood 82:2289.

EXPERIMENTAL DETAIL SECTION IV

Introduction

Non-Hodgkin lymphomas (NHL) represent one of the most commonmalignancies associated with human immunodeficiency virus (HIV)infection, and are recognized as an acquired immunodeficiency syndrome(AIDS)-defining condition (1-3). Since their initial observation in 1982(4), the incidence of AIDS-associated NHL (AIDS-NHL) has beenconsistently increasing (1, 2), and they now represent the most frequentHIV-associated malignancy in some AIDS risk groups, namely thehemophiliacs (5). Indeed, some estimates project that 10 to 20% of allnew NHL cases in the United States may eventually be related to AIDS(6).

AIDS-NHL are almost invariably B-cell derived NHL (1, 2, 7-12). Whencompared with NHL of similar histology arising in the immunocompetenthost, AIDS-NHL display distinctive clinical features, including latestage at presentation, poor prognosis, and the frequent involvement ofextranodal sites (1, 2, 7-12). Systemic AIDS-NHL are histologicallyheterogeneous, and have been initially classified into three distinctcategories, including small non cleaved cell lymphoma (SNCCL), large noncleaved cell lymphoma (LNCCL), and large cell-immunoblastic plasmacytoidlymphoma (LC-IBPL) (7, 9). Subsequently, most investigators have agreedto classify LNCCL and LC-IBPL as a single category under the term ofdiffuse large cell lymphoma (DLCL).

Some progress has been made in elucidating the molecular pathogenesis ofAIDS-SNCCL (1-3). AIDS-SNCCL is associated at variable frequency withmultiple genetic lesions, including Epstein Barr virus (EBV) infection,c-MYC translocation, RAS gene family mutation, and p53 inactiviation bypoint mutation and allelic loss (1, 3, 13-25). On the other hand, thepathogenesis of AIDS-DLCL is relatively less defined. EBV infectionappears to be the only genetic lesion associated with a significantfraction of these tumors, particularly with the subset displayingplasmacytoid features, p53 lesions have not been found and c-MYCactivation is restricted to a small minority of cases (1-3, 13-25).

Materials and Methods

Pathologic samples. Biopsy samples of lymph node, bone marrow,peripheral blood, or other involved organs from forty patients with AIDSwere collected during the course of standard diagnostic procedures.Thirty-two samples were derived from patients referred to the Departmentof Pathology, New York University, New York, N.Y. or to the Departmentof Pathology, Columbia University, New York, N.Y. Eight samples werederived from patients referred to the Departments of Hematology andPathology, University of Southern California School of Medicine, LosAngeles, Calif. Diagnosis was based on analysis of histopathology,immunophenotypic analysis of cell surface markers, and immunogenotypicanalysis of Immunoglobulin (Ig) gene rearrangement (32). In most cases,the fraction of malignant cells in the pathologic specimen was greaterthan 80%, as determined by cell suspension cytofluorometric or tissuesection immunohistochemical analysis of cell surface markers and by Iggene rearrangement analysis.

DNA extraction and Southern blot analysis. DNA was purified by digestionwith proteinase K, "salting out" extraction, and precipitation byethanol (33). For Southern blot analysis (34), 6 μg of DNA was digestedwith the appropriate restriction endonuclease, electrophoresed in a 0.8%agarose gel, denatured, neutralized, transferred to Duralon filters(Stratagene, LA Jolla, Calif.), and hybridized to probes which had been³² P-labeled by the random primer extension method (35). Filters werewashed in 0.2× SSC (NaCl/Na citrate)/0.5% sodium dodecyl sulphate (SDS)for 2 hours at 60° C. and then autoradiographed using intensifyingscreens (Quanta III; Dupont, Boston, Mass.).

DNA probes. Immunoglobulin gene rearrangement analysis was performedusing a J_(H) probe(36) (a gift of Dr. Korsmeyer) on HindIII, EcoRI, andBamHI digests. The organization of the BCL-6 locus was investigated byhybridization of XbaI, BazHI, and BglII digested DNA to the human BCL-6probe Sac4.0 (26-27). In selected cases, a second probe representativeof the BCL-6 locus, Sac0.8, was also used. The organization of the c-MYClocus was analyzed by hybridization of EcoRI and HindIII digested DNA tothe human c-MYC locus was analyzed by hybridization of EcoRI and HindIIIdigested DNA to the human c-MYC probe MC413RC, representative of thethird exon of the c-MYC gene (37). The presence of the EBV genome wasinvestigated with a probe specific for the EBV termini (5.2 KbBamHI-EcoRI fragment isolated from the fused BamHI terminal fragmentNJ-het) (38).

Experimental Results

Forty cases of systemic AIDS-NHL were studied, including 13 SNCCL and 24DLCL (8 LNCCL and 16 LC-IBPL). In addition, three cases of CD30+lymphomas, which have been sporadically reported in AIDS (39), were alsoincluded. All cases displayed a predominant monoclonal B-cell populationas determined by Ig gene rearrangement analysis.

Analysis of BCL-6 rearrangements. The BCL-6 gene contains at least 9exons spanning approximately 26 Kb of genomic DNA (27). Sequenceanalysis has shown that the first exon is non-coding and that thetranslation initiation codon is located within the third exon (27).Rearrangements of BCL-6 can be detected by Southern blot analysis usinga probe (Sac4.0) and restriction enzymes (BamHI and XbaI) which, incombination, explore a region of 15.2 Kb containing the 5' portion ofthe BCL-6 gene (27) (FIGS. 14A-14C). This same region was previouslyshown to contain the cluster of chromosomal breakpoints detected in NHLof the immunocompetent host (27, 29). Cases showing an abnormallymigrating band in only one digest were further studied by hybridizingthe Sac4.0 probe to additional digests (BglII) or, alternatively, byhybridizing BamHI and XbaI digests to a probe (Sac0.8) derived from theBCL-6 first intron, which, being located 3' of the breakpoint cluster,explores the reciprocal chromosome 3 (FIGS. 14A-14C). Only cases showingabnormally migrating bands with two restriction enzymes and/or twoprobes were scored as rearranged.

Rearrangements of BCL-6 were detected 5/24 AIDS-DLCL (20.8%), both inthe LNCCL (2/8; 25%) and in the LC-IBPL (3/16; 18.7%) variants (Table 3and FIGS. 14A-14C). All cases of AIDS-SNCCL and CD30+ lymphomasdisplayed a germline BCL-6 locus. The location of the breakpointsdetected in AIDS-HNL corresponds to the pattern most commonly observedin DLCL of the immunocompetent host.

                  TABLE 3    ______________________________________    Frequency of BCL-6 rearrangements in AIDS-NHL                         DLCL.sup.b    SNCCL.sup.a             LNNCL       LC-IBPL  CD30 + NHL.sup.c    ______________________________________    0/13     2/8         3/16     0/3    ______________________________________     .sup.a : SNCCL, small non cleaved cell lymphoma     .sup.b : DLCL, diffuse large cell lymphoma. The DLCL included in the pane     can be further distinguised into two subgroups (LNCCL, large non cleaved     cell lymphoma; and LCIBPL, large cell immunoblasticplasmacytoid lymphoma)     as previously reported (7,9).     .sup.c : NonHodgkin lymphoma expressing the CD30 cell surface antigen     (39).

Other genetic lesions. The other genetic lesions investigated in thepanel of AIDS-NHL included infection by EBV of the tumor clone,activation of the c-MYC and RAS proto-oncogenes, and inactivation of thep53 tumor suppressor gene. The experimental strategies used toinvestigate these lesions have been described in detail elsewhere (13,45, 40). For some of the cases, the molecular characterization of thesegenetic lesions have been previously reported (13, 14, 41); for theother cases, it has been assessed in the course of this study.

EBV infection was assessed by Southern blot hybridization using a proberepresentative of the EBV termini (38) which allows to analyze clonalityin EBV-infected tissues (23) (FIGS. 16A-16C). A monoclonal infection wasdetected in 5/13 (38%) SNCCL, 17/24 DLCL (71%) 3/8 (37.5%) LNCCL and14/16 (87.5%) LC-IBPL!, and 3/3 (100%) CD30+ cases.

Rearrangements of c-MYC were tested by hybridizing HindIII and EcoRIdigested DNAs with a probe representative of c-MYC exon 3(41) (FIGS.16A-16C). Rearrangements were present in 13/13 SNCCL (100%), 5/24(20.8%) DLCL 2/8 (25%) LNCCL and 3/16 (18.7%) LC-IBPL!, and 2/3CD30+cases.

Mutations of p53 and RAS were analyzed by a two step strategy. Singlestrand conformation polymorphism (SSCP) analysis was applied to p53exons 5 through 9 (in 29 cases) or p53 exons 5 through 8 (in 6 cases)and to N-, K-, and H-RAS exons 1 and 2 (in 29 cases); cases displayingan altered electrophoretic pattern by SSCP were further studied by DNAdirect sequencing of the PCR product. p53 mutations were scored in 8/13(61.5%) SNCCL, but in none of the DLCL tested (0/22). Finally, RASactivation by point mutation was positive in 3/13 (23%) SNCCL and in1/16 (6%) DLCL tested.

The molecular features of the cases displaying BCL-6 rearrangements arelisted in Table 4. Overall, BCL-6 rearrangements were detected both inthe presence and in the absence of clonal EBV infection of the tumor,whereas c-MYC alterations and p53 mutations were consistently absent inthe cases displaying BCL-6 rearrangements.

                  TABLE 4    ______________________________________    Molecular features of AIDS-DLCL.sup.a                    CLON-    PATIENT           HISTOL..sup.b                    ALITY   BCL-6 EBV  c-MYC p53  RAS    ______________________________________    DK782  LNCCL    +       +     -    -     -    -    DK1178 LNCCL    +       +     -    -     -    -    DK1028 LNCCL    +       -     -    -     -    -    DK3973 LNCCL    +       -     +    -     -    -    DK773  LNCCL    +       -     -    +     -    -    RDF834 LNCCL    +       -     +    +     -    -    DK1452 LNCCL    +       -     -    -     -    -    DK64   LNCCL    +       -     +    -     -    +    DK771  LC-IBPL  +       +     +    -     -    -    K827   LC-IBPL  +       +     +    -     -    -    DS16   LC-IBPL  +       +     +    -     -    ND    DK3537 LC-IBPL  +       -     +    +     -    -    DK3357 LC-IBPL  +       -     +    -     -    -    DK63   LC-IBPL  +       -     +    -     -    -    DK1446 LC-IBPL  +       -     +    +     -    -    DK3479 LC-IBPL  +       -     -    -     -    -    DK2092 LC-IBPL  +       -     +    -     -    -    DS17   LC-IBPL  +       -     -    -     -    ND    DS45   LC-IBPL  ND      -     +    -     -    ND    DS46   LC-IBPL  +       -     +    +     -    ND    DS93   LC-IBPL  +       -     +    -     -    ND    DS136  LC-IBPL  +       -     +    -     -    ND    DS155  LC-IBPL  +       -     +    -     -    ND    DS165  LC-IBPL  +       -     +    -     -    ND    ______________________________________     .sup.a : The results of the analysis of EBV, cMYC, p53 and RAS of some of     these cases have been previously reported (14, 15, 41).     .sup.b : LNCCL, large non cleaved cell lymphoma; LCIBPL, large     cellimmunoblastic plasmacytoid lymphoma     .sup.c : ND, not done

Experimental Discussion

Diffuse large cell lymphoma (DLCL) represents the most frequent type ofAIDS-NHL in the HIV-infected adult (8). Despite its epidemiologicrelevance, the molecular pathogenesis of these tumors is largelyunclarified (3). Analysis of the genomic configuration of BCL-6 in apanel of AIDS-NHL indicates that BCL-6 rearrangements are involved inapproximately 20% of AIDS-DLCL, whereas they are consistently negativein AIDS-SNCCL. In this respect, BCL-6 rearrangements may be consideredthe first identified genetic lesion specific for the DLCL type amongAIDS-NHL. BCL-6 rearrangements are present in both subgroups of DLCL,i.e. LNCCL and LC-IBPL, and occur both in the absence and in thepresence of EBV infection of the tumor clone (Table 4). On the otherhand, BC.L-6 rearrangements were never detected in AIDS-DLCL carryingc-MYC alterations (Table 4).

The molecular pathway leading to AIDS-SNCCL involves c-MYCrearrangements, p53 mutations, and EBV infection in 100%, 60%, and 40%of the cases, respectively (13-26). The presence of somatichypermutation in the immunoglobulin variable regions utilized byAIDS-SNCCL points to chronic antigen stimulation as an additionalmechanism in the development of these tumors. The second genetic pathwayis associated with AIDS-DLCL, involves EBV in the large majority ofcases, as well as c-MYC and/or BCL-6 rearrangements in a fraction ofcases (13-26). These distinct pathogenetic mechanisms correlate with anumber of clinical features which distinguish AIDS-SNCCL from AIDS-DLCL,including different age of onset and different CD4 counts at the time oflymphoma development (1,2,8). Results suggest that the frequency ofBCL-6 rearrangements in AIDS-DLCL is significantly lower than that inDLCL in the immunocompetent host, where BCL-6 rearrangements occur inmore than 40% of the cases. It is possible that the genetic pathogenesisof these two groups of tumors is different, and that the molecularmechanisms active in AIDS-DLCL are characterized by a higher degree ofheterogeneity. Among DLCL in the immunocompetent host, BCL-6rearrangements are associated with distinct clinical features, includingthe extranodal origin of the lymphoma and the lack of bone marrowinvolvement. In addition, the presence of this rearrangement appears torepresent a favorable prognostic marker.

REFERENCES FOR SECTION IV

1. Karp, J. E., and Broder, S. (1991) Acquired Immunodeficiency Syndromeand non-Hodgkin's lymphomas, Cancer Res. 51:4743.

2. Levine, A. M. (1992) Acquired Immunodeficiency Syndrome-relatedlymphoma, Blood 80:8.

3. Gaidano, G., and Dalla-Favera, R. (1992) Biologic aspects of humanimmunodeficiency virus-related lymphoma, Curr. Opinion Oncol. 4:900.

4. Ziegler, J. L., et al. (1982) Outbreak of Burkitt's like lymphoma inhomosexual men, Lancet 2:631.

5. Ragni, M. V., et al. (1993) Acquired immunodeficiencysyndrome-associated non-Hodgkin's lymphomas and other malignancies inpatients with hemophilia, Blood 81:1889.

6. Gail, M. H., et al. (1991) Projection of the incidence ofnon-Hodgkin's lymphoma related to acquired immunodeficiency syndrome, J.Natl. Cancer Inst. 83:965.

7. Raphael, B. G., and Knowles, D. M. (1990) Acquired. immunodeficiencysyndrome-associated lymphomas, Sermin. Oncol. 17:361.

8. Beral, V., et al. (1991) AIDS-associated non-Hodgkin lymphoma, Lancet337:805.

9. Knowles, D. M., et al. (1988) Lymphoid neoplasia associated with theacquired immunodeficiency syndrome (AIDS), Ann. Int. Med. 108:744.

10. Levine, A. M., et al. (1984) Development of B-cell lymphoma inhomosexual men, Ann. Intern. Med. 100:7.

11. Carbone, A., et al. (1991) A clinicopathologic study of lymphoidneoplasias associated with human immunodeficiency virus infection inItaly, Cancer 68:842.

12. Ioachim, H. L., et al. (1991) Acquired immunodeficiencysyndrome-associated lymphomas:

Clinical, pathologic, immunologic, and viral characteristics of 111cases, Hum. Pathol. 22:659.

13. Ballerini, P., et al. (1993) Multiple genetic lesions in acquiredimmunodeficiency syndrome-related non-Hodgkin's lymphoma, Blood 81:166.

14. Gaidano, G., et al. (1993) In vitro establishment of AIDS-relatedlymphoma cell lines: phenotypic characterization, oncogene and tumorsuppressor gene lesions, and heterogeneity in Epstein-Barr virusinfection, Leukemia 7:1621.

15. Groopman, J. E., et al. (1986) Pathogenesis of B-cell lymphoma in apatient with AIDS, Blood 67:612.

16. Pelicci, P.-G., et al. (1986) Multiple monoclonal B cell expansionsand c-myc oncogene rearrangements in acquired immune deficiencysyndrome-related lymphoproliferative disorders. Implications forlymphomagenesis, J. Exp. Med. 164:2049.

17. Subar, M., et al. (1988) Frequent c-myc oncogene activation andinfrequent presence of Epstein-Barr Virus genome in AIDS-associatedlymphoma, Blood 72:667.

18. Haluska, F. G., et al. (1989) Molecular resemblance of anAIDS-associated lymphoma and endemic Burkitt lymphomas: implications fortheir pathogenesis, Proc. Natl. Acad. Sci. USA 86:8907.

19. Meeker, T. C., et al. (1991) Evidence for molecular subtypes ofHIV-associated lymphoma: division into peripheral monoclonal, polyclonaland central nervous system lymphoma, AIDS 5:669.

20. Epstein-Barr virus and AIDS associated lymphomas. Editorial, Lancet338:979, (1991).

21. Hamilton-Dutoit, S. J., et al. (1991) Detection of Epstein-Barrvirus genomes in AIDS related lymphomas: sensitivity and specificty ofin situ hybridization compared with Southern blotting, J. Clin. Pathol.44:676.

22. Hamilton-Dutoit, S. J., et al. (1991) AIDS-related lymphoma.Histopathology, immunophenotype, and association with Epstein-Barr virusas demonstrated by in situ nucleic acid hybridization, Am. J. Pathol.138:149.

23. Neri, A., et al. (1991) Epstein-Barr virus infection precedes clonalexpansion in Burkitt's and acquired immunodeficiency syndrome-associatedlymphoma, Blood 77:1092.

24. Nakamura, H., et al. (1993) Mutation and protein expression of p53in acquired immunodeficiency syndrome-related lymphomas, Blood 82:920.

25. Carbone, A., et al. (1993) Human immunodeficiency virus-associatedsystemic lymphomas may be subdivided into two main groups according toEpstein-Barr viral latent gene expression, J. Clin. Oncol. 1:1674.

26. Ye, B. H., et al. (1993) Cloning of BCL-6, the locus involved inchromosome translocations affecting band 3q27 in B-cell lymphoma, CancerRes. 53:2732.

27. Ye, B. H., et al. (1993) Alterations of a zinc finger-encoding gene,BCL-6, in diffuse large cell-lymphoma, Science 262:747.

28. Baron, B. W., et al. (1993) Identification of the gene associatedwith the recurring chromosomal translocations t(3;14)(q27;q32) andt(3;22) (q27;q11) in B-cell lymphomas, Proc. Natl. Acad. Sci. USA90:5262.

29. Kerckaert, J.-P., et al. (1993) LAZ3, a novel zinc-finger encodinggene, is disrupted by recurring chromosome 3q27 translocations in humanlymphoma, Nature Genet. 5:66.

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EXPERIMENTAL DETAIL SECTION V

Introduction

The group of diffuse lymphomas with a large cell component (DLLC),including diffuse mixed, immunoblastic, and large cell subtypes, and thegroup of follicular lymphomas, each comprise about 40 per cent ofnon-Hodgkin's lymphomas (NHL) in this country (1). Together, theincidence of NHL is increasing at 3 to 4 per cont a year, a rate secondonly to that of malignant melanoma and lung cancer in women (2). Despitesignificant advances in treatment, approximately half of patients withDLLC will succomb to their disease, although "high risk" individuals maysuccessfully be treated by intensive chemotherapy and radiotherapyregimens including autologous bone marrow transplantation (3-7). Theformulation of prognostic models allow clinical trials to be directedtoward groups of patients with different risks for failure afterconventional treatment (5).

Cytogenetic studies as well as molecular genetic analysis of alterationsinvolving proto-oncogenes and tumor suppressor genes have providedinsights into the pathogenesis of NHL, and have also contributeddiagnostic and prognostic markers (8,9). Examples include rearrangementsof the BCL-2 gene at 18q21 observed in up to 85 per cent of follicularlymphomas, the BCL-1 gene at 11q13 rearranged in intermediatedifferentiation NHL, and the MYC gene, perturbed in Burkitt's lymphoma(8,9). While no recurring genetic abnormality has been specificallyassociated with diffuse large cell lymphoma, rearrangement of BCL-2 hasbeen observed in 20 to 30 per cent of cases, where it has beenassociated with decreased overall or disease free survival (10-12).Chromosomal translocations including those involving the MYCproto-oncogene, while noted in DLLC, were not as prognosticallysignificant as other recurring chromosomal abnormalities (8,13).

BCL-6 (14-19) rearrangement is found to denote a subset of DLLCcharacterized by extranodal presentation and a favorable clinicaloutcome. These results indicate that, in concert with other clinicalfeatures, this molecular marker may be utilized as a prognosticindicator at the time of diagnosis.

Materials and Methods

This study was comprised of 102 cases of DLLC studied at diagnosis withdocumented clonal rearrangement of the IGH gene and DNA available forfurther analysis, derived from 229 DLLC serially ascertained over a nineyear period. Excluded were 127 cases studied at relapse, T cell DLLC, orcases for which no DNA was available. For this study, DLLC was definedas lymphomas of diffuse large cleaved, non-cleaved, immunoblastic, ormixed subtype, according to the International Working Formulation (20)as classified by a hematopathologist (DCS or DF). Cytogenetic analysiswas attempted on each of the specimens as previously described (21). Fordetection of BCL-6 rearrangements, DNA from each case was digested withBamHI and XbaI and subjected to Southern blot analysis utilizing a 4 kbSac1-Sac1 fragment of the BCL-6 gene as a probe (19). Cases which didnot yield metaphases for karyotypic analysis were also analyzed forrearrangement of the MBR and MCR breakpoint regions of the BCL-2 gene,as previously described (11). Aggregate descriptions of 47 of the casesin the current series were included in prior reports of cytogeneticabnormalities in DLLC (11, 13, 14). A detailed molecular analysis of 8cases (nos. 352, 755, 1098, 1254, 1403, 1444, 1445) demonstrating BCL-6rearrangement has been reported separately (19).

For each case, clinical data were compiled as previously described (22).Stage was assessed according to the modified Ann Arbor criteria (25).For the purposes of separate evaluation of number of extranodal sites ofdisease, radiographs or pathologic involvement of these sites werescored. In the quantitation of extranodal sites of disease as aprognostic variable, bone marrow, but not splenic involvement wasscored, in accord with the International Prognostic Index (5).

Clinical endpoints including complete response and freedom fromprogression were defined as previously described (3). Of 102 patientswith DLLC genetically analyzed prior to cytotoxic treatments, 93received systemic chemotherapy. Nine patients with early stage diseasewere treated by surgical resection and/or radiation therapy. Allpatients were treated with curative intent. Chemotherapy treatments wereclassified into three groups: NHL-4, CHOP and BACOP (1st generation);m-BACOD, NHL-7 (2nd generation); MBCOP-B, NHL-9, NHL-14, NHL-15, L-20(3rd generation)(4,24,29). Eight patients expired before completion oftherapy, with incomplete staging evaluations, or of infectiouscomplications during or shortly after treatment. These cases wereconsidered not valuable for the determination of remission status, butwere included in the analysis of overall survival and freedom fromprogression. One patient was judged to be a complete remission which wasconfirmed by autopsy after expiration due to infectious complications 3weeks after completion of protocol treatment. All deaths, regardless ofcause were considered as endpoints in the analysis of overall survival.Median survival was determined by the method of Kaplan and Meler (30).Analysis of correlations between gene rearrangements and clinicalfeatures were performed utilizing Fisher's exact test (13). Means werecompared utilizing two sample t-tests. Univariate comparisons ofsurvival and duration free from progression were made by log rank test.Survival and freedom from progression estimates are quoted withconfidence intervals (CI) given in parentheses. Multivariate analysiswas performed utilizing the Cox regression model (31). Stepwise multiplelogistic regression was sued in the multivariate analysis of factorsprognostic for achieving a complete response. For all statisticalanalyses, a P<0.05 based on a 2-sided test was considered significant.

Experimental Results

Of 102 cases of DLLC studied at diagnosis, 23 demonstrated BCL-6rearrangement, 21 demonstrated t(14;18) or rearrangement of BCL-2, and58 demonstrated no evidence of either BCL-6 or BCL-2 rearrangement.Representative results of hybridization analysis for rearrangement ofBCL-6 are depicted in FIGS. 17A-17B. The clinical characteristics ofgroups according to BCL-6 or BCL-2 rearrangement are summarized in Table5. The histologic subtypes and clinical features of the BCL-6 rearrangedcases are shown in Table 6.

The key to Table 6 is as follows: Underlining signifies site from whichbiopsy was performed. Histology: DLC=diffuse large cell;IMB-immunoblastic; DML =diffuse mixed lymphoma; LDH=lactatedehydrogenase in units/ml; (B)=bulky disease (>8 cm or 1/3 thoracicdiameter); CHOP=cyclophosphamide, daunorubicin, vincristine, prednisone;MACOPB=methotrexate, daunorubicin, cyclophosphamide, vincristine,prednisone, bleomycin; MBACOD=same drugs as MACOPB with dexamethasoneinstead of prednixone and drugs in different schedule; PrCyBom-drugs ofMACOPB plus cytosine arabinoside, etopiside, methotrexate;L-20-vincristine, cyclophosphamide, methotrexate, daunorubicin,prednisone, cytosine arabinoside, 1-asparaginase, BCNU,6-mercaptopurine, dactinomycin; 1-20 includes randomization toautologous transplantation; NHL-7=CHOP plus methyl GAG, etoposide(36),NHL-14=short course PrCyBom; NHL-15=high dose daunorubicin, vincristine,cyclophosphamide(29); RT=radiation therapy; SURG=surgery CR=completeresponse; sCR=surgical complete response (all evaluable diseaseresected); PR=partial response; "+"=alive at last follow-up; e=expired;rel=relapse; NE=not evaluable; *=Patient 1445 had a history of low gradeNHL of eyelid 7 years earlier, treated by radiotherapy. # Skininvolvement of patient 252 was not noted in a prior report (14) andpatient 1445 had a history of low grade NHL of eyelid 7 years earlier,treated by radiotherapy.

While each of the BCL-6 rearranged cases was classified as a DLLC, therange of morphologies included diffuse large cell (cleaved andnon-cleaved), and less frequently, immunoblastic, or mixed histologies.Extensive necrosis and extranodal extension were common histologicfeatures, and were present in one of two cases of BCL-6 rearrangementwhich did not show clinical evidence of extranodal disease.

The BCL-6 rearranged cases had a mean age of 64.1 years at presentationand a high frequency of extranodal involvement by disease; 19 of 23cases had stages IE, IIE, IIIE or stage IV desease, compared to 48 of 79of BCL-6 germline cases (p+0.07). Extranodal sites included muscle orsubcutaneous tissues (6 cases), stomach (5 cases), lung or pleura (5cases), skin, breast, bowel, thyroid, pancreas, or kidney, as assessedby biopsy or radiographic abnormalities which improved afterchemotherapy. Of the 7 cases with state IE or IIE decrease, 5 wereprimary extranodal lymphomas, while 2 were extranodal extensions from aprimary nodal site. Two cases were primary splenic lymphomas. In twocases, there was only peripheral adenopathy. Compared to BCL-6 germlinecases, there was no significant difference in the proportion of BCL-6rearranged cases with stage IV disease. Bone marrow involvement wasobserved in 15 of 75 BCL-6 germline cases biopsied, compared to only 1of the 23 stage IV BCL-6 rearranged cases (P=0.1).

All but one of the 23 patients with BCL-6 rearrangement at the time ofdiagnosis received anthracycline-containing chemotherapy. This patientremained free of disease eight years after resection of a primarysplenic large cell lymphoma. At median follow-up in excess of two years,21 of the 23 patients with BCL-6 rearrangement survived; the actuarialsurvival was 91 per cent (CI 80 per cent to 100 per cent). Two patientsexpired during or immediately following treatment; an autopsy in onecase revealed no evidence of lymphoma. This patient, and 19 others werejudged to have achieved a complete remission after treatment. Twopatients relapsed with recurrent disease in the lung and two patientshad persistent subcutaneous masses. One of the relapse patients (case295) went on to autologous transplanation and remains free of disease 78months post-transplant.

With respect to known prognostic variables, the proportion of the BCL-6rearranged cohort with LDH>500 U per liter was similar to the proportionof the BCL-6 germline DLLC (3/23 versus 13/79; P=0.99). Five of 23 casesof DLLC with BCL-6 rearrangement demonstrated bulky disease, compared to35 of 79 cases without BCL-6 rearrangement (P=0.1). The proportion ofcases with "limited stage" (I, IE, II, or IIE) disease was comparable inthe cohorts with and without BCL-6 rearrangement (Table 5).

                  TABLE 5    ______________________________________    Characteristics of 102 cases of DLLC                       BCL-6-     BCL-6-              BCL-6+   BCL2-      BCL2+    ______________________________________    n=          23         58         21    Mean age (years)                 64.1       52.7       62.8    Mean lactate                405        331        389    dehydrogenase (U/ml)    Mean extranodal sites                  1.6         .93       .81    Bone marrow 1/23       8/54       7/21    involvement    Stage    I(IE)       3(1)       3(3)       0    II(IIE)     7(6)       22(6)      5    III(IIIE)   2(1)       8(2)       4    IV          11         25         12    Histology    Diffuse large cell                20         53         19    Diffuse mixed                 1          2         1    Immunoblastic                 2          3         1    Treatment    1st generation chemo.                12         16         10    2nd generation chemo.                 1         10         3    3rd generation chemo.                 9         24         8    other        1          8         0    Complete Remission                20/23      35/50      15/21    Rate        86%        70%        71%    Projected survival                91%        59%        46%    at 36 months                (CI        (CI        (CI                80%-100%)  44%-74%)   21%-72%)    Projected freedom                82%        56%        31%    from progression                (CI        (CI        (CI    at 36 months                66%-98%)   43%-70%)   8%-53%)    ______________________________________

                                      TABLE 6    __________________________________________________________________________    Clinical features of 23 cases of DLLC with BCL6 rearrangement    CASE AGE/   EXTRANODAL      LDH         CLINICAL    NUMBER         SEX            STAGE                SITES    HISTOLOGY                                (BULK)                                    TREATMENT                                            OUTCOME    __________________________________________________________________________    102  66/F            IIS spleen   DLC    3624                                    SURG,CHOP                                            CR,96+    147  61/F            IS  spleen   DLC    365 SURG,RT sCR,101+                                (B)    252  54/M            IV  spleen,skin                         DLC    126 MACOPB  CR,58+    278  68/M            IV  pleura,  DLC    235 MACOPB  PR,6e                iliac mass      (B)    295  46/M            IV  lung     DLC    179 MACOPB,L-20                                            CR,rel,97+    352  53/F            IV  stomach,liver,                         IMB    775 MACOPB  CR,81+                spleen,small    (B)                bowel,pleural                effasion    470  74/F            IV  lung     DLC    224 CHOP    CR,30+    534  70/F            IIES                spleen,mass                         DLC    278 CHOP    CR,80+                involving pancreas                                (B)    763  79/F            IIE stomach  DLC    196 CHOP    CR,60+    970  75/M            IV  kidney,  DLC    240 NHL-14  CR,4e                stomach    1020 60/M            IIIE                tonsil,  DLC    303 CHOP    CR,100+                pancreas    1056 63/M            IIE stomach  DLC    213 SURG,MBACOD                                            CR,100+    1058 59/M            IIE axillary pass                         DLC    206 PrCyBom CR,37+                involving breast    1098 74/F            IV  subcutaneous                         DML    181 RT,CHOP PR,36+                masses    1189 71/M            IV  subcutaneous                         DLC    330 CHOP    PR,21+                masses    1254 74/F            IIE thyroid  DLC    196 CHOP/RT CR,27+    1264 76/F            IV  Lung,liver                         DLC    234 CHOP    CR,rel,27+                spleen,kidney    1299 50/M            IE  deltoid mass,                         DLC    529 PrCyBom CR,16+                bone            (B)    1363 47/M            III NONE     DLC    129 NHL-15  CR,16+    1403 62/M            I   NONE     IMB    222 CHOP,RT CR,11+    1407 71/M            IIE stomach  DLC    206 SURG,CHOP                                            CR,12+    1444 70/F            IV  lung     DLC    150 CHOP    CR,14+    1445 63/F            IV  neck mass                         DLC    174 NHL-15  CR,8+                involving                muscle*,bone                marrow    __________________________________________________________________________

Multivariate analysts of clinical outcome. The median duration free fromprogression of disease was not reached in the BCL-6 rearranged DLLCcompared to 70 months for BCL-6 germline cases, regardless of BCL-2status (P=0.009) (FIG. 18A). Projected freedom from progression at 36months was 82% (CI 66%-98%) and 49% (CI 37%-60%), respectively.Multivariate analysis revealed that four variables, BCL-6 status, stageIV disease, bulk of disease, and LDH (log transformed) were the mostpowerful prognostic indicators for freedom from progression (Table 7).Multivariate analysis of survival demonstrated that bulk, LDH, BCL-6status, and stage IV disease were the most useful predictors of overallsurvival (P=0.01, P=0.02, P=0.02, P=0.05, respectively).

                  TABLE 7    ______________________________________    Multivariate analysis of freedom from progression    Variables selected    into Cox regression                   Relative    P value    model          Risk        (Wald chi square)    ______________________________________    BCL-6 rearranged                   0.18 (CI) .04-.78)                               0.007    Bulky disease  2.4 (CI 1.3-7.4)                               0.01    Stage IV disease                   2.1 (CI 0.98-5.2)                               0.03    LDH (log transformed)                   1.6 (CI 1.1-3.9)                               0.05    ______________________________________

The prognostic value of BCL-6 gene status was compared to risk variablescalculated according to the International Prognostic Index⁵, includingserum LDH level, stage, performance status, and number of extranodalsites. A cox regression analysis confirmed the independent prognosticvalue of BCL-6 gene status;

patients with BCL-6 rearrangement had a relative risk (RR) of dying of0.09 (CI 0.02 to 0.42) compared to patients without BCL-6 rearrangement,controlling for the other prognostic variables in the model (P=0.002).

When cases were considered with respect to BCL-2 status, the BCL-2rearranged cases demonstrated a trend for a decreased survival comparedto BCL-2 germline cases, regardless of BCL-6 status (P=0.12). When BCL-6and BCL-2 status were considered together (FIG. 18B), BCL-6 rearrangedcases demonstrated a projected actuarial survival at 36 months of 91%(CI 80%-100%) compared to 59% (CI 44%-74%) for the BCL-6 germline/BCL-2germline cohort, and 46% (CI 21%-72%) for the BCL-2 rearranged cohort.While the logrank test between these three cohorts demonstrated adifference in survival (P=0.02, FIG. 18B), the major factor driving thesignificant summary P value was the better survival of the BCL-6rearranged cohort. The projected freedom from progression at 36 monthswas 82% (CI 66%-98%), 56% (CI 43%-70%) and 31% (CI 8%-53%) for the threegroups. Median follow-up for survivors was two years. BCL-2rearrangement did not emerge as an independent prognostic marker in themultivariate analysis of survival or freedom from progression.

There was also no prognostically significant effect of generation ofchemotherapy treatment on survival, or freedom from progression (P=0.95,0.21, respectively). There was a trend for a higher complete responserate among the BCL-6 rearranged cohort (Table 5, P=0.1), althoughlogistic regression revealed that only the clinical parameters LDH,stage IV, and bulk of disease were independent predictors of response.

Relationship between BCL-6, BCL-2, and 8q24 rearrangements. Of the 79cases which lacked BCL-6 rearrangement, 21 demonstrated t(14;18)(q32;q21) or rearrangement of BCL-2 by molecular analysis. These caseswere characterized by an older age at diagnosis, but were similar to thelarger cohort of BCL-2 negative, BCL-6 negative cases with respect toLDH, and distribution of histologies (Table 6).

Nine cases of DLLC demonstrated t(8;14) (q24;q32). Three of thesebiopsies were from extranodal sites including liver, bone and softtissue. Two additional cases were splenic lymphomas. In two cases,t(8;14) bearing DLLC also demonstrated BCL-6 rearrangement. There was noimpact on survival of the t(8;14) in DLLC with or without BCL-6rearrangement. The two cases of t(8;14) with co-incident BCL-6rearrangement did not show evidence of histologic transformation orother unusual histologic features. One of these cases (no. 147) was thesingle case treated by splenectomy and radiation therapy alone. Thesecond case was the single BCL-6 rearranged case successfully salvagedby autologous transplantation.

Cytogenetic features, including the relationship between 3q27 and BCL-6rearrangement. Of the 65 DLLC with karyotypic abnormalities, 14demonstrated translocations and one a deletion affecting band 3q27; only11 among these 15 cases showed rearrangement of BCL-6 Five cases withapparently normal chromosomes 3 demonstrated BCL-6 rearrangements by DNAanalysis.

Experimental Discussion

As a group, DLLC are among the most common forms of NHL seen in thiscountry (1). These tumors have not, however, been associated with acharacteristic genetic abnormality (8). Seen in the vast majority offollicular lymphomas, t(14;18) (q32;q21) or its molecular equivalent,BCL-2 rearrangement, have been observed in 20 to 30 per cent of DLLC(8). In such cases, the t(14;18) may reflect a follicular origin ofthese tumors. The recognition of translocations involving 3q27 and thesites of IG genes, 14q32, 22q11, and 2p12, in predominantly diffuse NHLled to the molecular cloning of BCL-6 (14-19). While not unique todiffuse large cell lymphomas, translocations affecting 3q27 wereobserved only in 7 of>200 cases of follicular NHL with abnormalkaryotype reported in catalog of chromosome abnormalities in cancer(32). Of 28 cases of follicular NHL analyzed in a prior study, nonedemonstrated rearrangement of BCL-6 (19). BCL-6 rearrangement wasestablished as the most common genetic lesion specific to DLLC at thetime of diagnosis.

Unlike 18q21 translocations in NHL, which to date only have involved IGgene loci as reciprocal partners, 3q27 translocations demonstrated amarked promiscuity of rearrangement partners. In addition to the sitesof the IG genes, reciprocal translocations involving the 3q27-29 regionwith at least 12 other loci; a total of 79 DLLC with 3q27 translocationshas been demonstrated.

Since 4 tumors in the current series with documented 3q27 aberrationsdid not reveal BCL-6 rearrangement with the probe used in this study,the true frequency of BCL-6 rearrangement in DLLC at diagnosis may behigher than the 23 per cent rate reported here. Additional breakpointsmay be documented outside the recognized break cluster region of BCL-6(19), in neighboring genes such as EV-1 (34), or in other genes not yetdescribed. Such molecular heterogeneity is not unique in NHL; seeminglyidentical chromosomal translocations have been shown to demonstrate adiversity of breakpoints possibly involving different genes (35).

The frequent occurrence of BCL-6 rearrangement in DLLC characterized byextranodal involvement represents one of the few genetic markers forthis subset of lymphoma (8). Rearrangements of BCL-1, BCL-2, or BCL-3have been documented infrequently in extranodal lymphomas (36-38), while5 of 12 gastric lymphomas in one series demonstrated MYC (8q24)rearrangement (38). The current series did not confirm the associationbetween 8q24 rearrangement and gastric lymphoma, although t(8;14) wasseen in five cases of extranodal lymphoma, one of which also showedBCL-6 rearrangement. The proportion of BCL-6 rearranged cases withstages IE, IIE, IIIE, or IV disease was higher than the proportion ofBCL-6 germline DLLC; in the latter group, stage IV disease was morecommonly due to bone marrow involvement. Whether this association withextranodal involvement of disease reflects an effect of the primaryderegulation of BCL-6 or "secondary" genetic events associated withtumor progression (8,21) is unclear. The observation of t(3;22), t(2;3),or t(3;14) as solitary cytogenetic abnormalities in some tumors (14,15),is consistent with a primary pathogenetic role for this translocation.

While this analysis and two other reports did not confirm the very shortsurvival of BCL-2 rearranged DLLC initially reported (10, 12, 13, 39),the BCL-2 rearranged DLLC did demonstrate a trend for a poorer overallsurvival. The finding of a favorable prognosis for the subset of stageIE-IIE extranodal DLLC with BCL-6 rearrangement is consistent with priorreports of a good prognosis associated with localized extranodal largecell NHL treated with surgery or radiotherapy (40). Extranodalinvolvement in advanced stage disease, noted in the majority of theBCL-6 rearranged cases, has generally been considered a poor prognosticfactor in large series of DLLC, although the negative impact of thisfeature was most evident when combined with other adverse indicatorssuch as bulk, high LDH, or low performance status (5, 22, 41). Incontrast, bone marrow involvement, observed in 22 percent of DLLC, andconsidered an extranodal site in the International Prognostic Index (5),was rare in the BCL-6 rearranged cohort. The favorable treatment outcomeof the BCL-6 cohort, must also be tempered by the observation of relapseor residual disease in 3 of the patients still alive. An additionalrelapse case remains in remission 6 years after "salvage" autologoustransplantation.

The BCL-6 rearranged cohort of DLLC also possessed other clinicalmarkers of favorable prognosis; although comparable with respect to LDHand proportion with stage I-IIIE disease, the proportion of cases withbulky disease or bone marrow involvement was lower in the BCL-6rearranged cohort. Multivariate analysis suggested, however, that BCL-6gene rearrangement added independent prognostic power when analyzedtogether with clinically-derived variables of the InternationalPrognostic Index (5). This observation is illustrated by case 352, whichdisplayed both BCL-6 rearrangement as well as clinical featuresconsistent with a high level of risk in the International Index(elevated LDH, extensive extranodal involvement, low performance status,and stage IV disease), but who attained a durable remission.

Because of issues of toxicity versus efficacy of autologous bone marrowtransplantation or peripheral stem cell rescue, the identification ofboth favorable and unfavorable prognostic markers offers the potentialto stratify treatment approaches to DLLC based on risk groups (4-7, 22,41). The probability of treatment failure remains as high as 25-40 percent for the most favorable subsets of DLLC based on current prognosticmodels, highlighting the need for genetic or other prognostic markers(5). In addition to its potential diagnostic and prognosticapplications, the further identification of BCL-6 breakpoint regionsoffers the opportunity to develop new polymerase chain reaction-derivedmeasures of minimal residual disease (43). The availability of BCL-6rearrangement as a new molecular marker of large cell lymphomaconstitutes a potentially important clinical tool in the management ofpatients with this desease.

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    __________________________________________________________________________    SEQUENCE LISTING    (1) GENERAL INFORMATION:    (iii) NUMBER OF SEQUENCES: 9    (2) INFORMATION FOR SEQ ID NO:1:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 3720 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 328..2445    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:    GGCCCCTCGAGCCTCGAACCGGAACCTCCAAATCCGAGACGCTCTGCTTATGAGGACCTC60    GAAATATGCCGGCCAGTGAAAAAATCTTATGGCTTTGAGGGCTTTTGGTTGGCCAGGGGC120    AGTAAAAATCTCGGAGAGCTGACACCAAGTCCTCCCCTGCCACGTAGCAGTGGTAAAGTC180    CGAAGCTCAAATTCCGAGAATTGAGCTCTGTTGATTCTTAGAACTGGGGTTCTTAGAAGT240    GGTGATGCAAGAAGTTTCTAGGAAAGGCCGGACACCAGGTTTTGAGCAAAATTTTGGACT300    GTGAAGCAAGGCATTGGTGAAGACAAAATGGCCTCGCCGGCTGACAGCTGT351    MetAlaSerProAlaAspSerCys    15    ATCCAGTTCACCCGCCATGCCAGGGATGTTCTTCTCAACCTTAATCGT399    IleGlnPheThrArgHisAlaArgAspValLeuLeuAsnLeuAsnArg    101520    CTCCGGAGTCGAGACATCTTGACTGATGTTGTCATTGTTGTGAGCCGT447    LeuArgSerArgAspIleLeuThrAspValValIleValValSerArg    25303540    GAGCAGTTTAGAGCCCATAAAACGGTCCTCATGGCCTGGAGAGGCCTG495    GluGlnPheArgAlaHisLysThrValLeuMetAlaTrpArgGlyLeu    455055    TTCTATAGCATCTTTACAGACCAGTTGAAATGCAACCTTAGTGTGATC543    PheTyrSerIlePheThrAspGlnLeuLysCysAsnLeuSerValIle    606570    AATCTAGATCCTGAGATCAACCCTGAGGGATTCTGCATCCTCCTGGAC591    AsnLeuAspProGluIleAsnProGluGlyPheCysIleLeuLeuAsp    758085    TTCATGTACACATCTCGGCTCAATTTGCGGGAGGGCAACATCATGGCT639    PheMetTyrThrSerArgLeuAsnLeuArgGluGlyAsnIleMetAla    9095100    GTGATGGCCACGGCTATGTACCTGCAGATGGAGCATGTTGTGGACACT687    ValMetAlaThrAlaMetTyrLeuGlnMetGluHisValValAspThr    105110115120    TGCCGGAAGTTTATTAAGGCCAGTGAAGCAGAGATGGTTTCTGCCATC735    CysArgLysPheIleLysAlaSerGluAlaGluMetValSerAlaIle    125130135    AAGCCTCCTCGTGAAGAGTTCCTCAACAGCCGGATGCTGATGCCCCAA783    LysProProArgGluGluPheLeuAsnSerArgMetLeuMetProGln    140145150    GACATCATGGCCTATCGGGGTCGTGAGGTGGTGGAGAACAACCTGCCA831    AspIleMetAlaTyrArgGlyArgGluValValGluAsnAsnLeuPro    155160165    CTGAGGAGCGCCCCTGGGTGTGAGAGCAGAGCCTTTGCCCCCAGCCTG879    LeuArgSerAlaProGlyCysGluSerArgAlaPheAlaProSerLeu    170175180    TACAGTGGCCTGTCCACACCGCCAGCCTCTTATTCCATGTACAGCCAC927    TyrSerGlyLeuSerThrProProAlaSerTyrSerMetTyrSerHis    185190195200    CTCCCTGTCAGCAGCCTCCTCTTCTCCGATGAGGAGTTTCGGGATGTC975    LeuProValSerSerLeuLeuPheSerAspGluGluPheArgAspVal    205210215    CGGATGCCTGTGGCCAACCCCTTCCCCAAGGAGCGGGCACTCCCATGT1023    ArgMetProValAlaAsnProPheProLysGluArgAlaLeuProCys    220225230    GATAGTGCCAGGCCAGTCCCTGGTGAGTACAGCCGGCCGACTTTGGAG1071    AspSerAlaArgProValProGlyGluTyrSerArgProThrLeuGlu    235240245    GTGTCCCCCAATGTGTGCCACAGCAATATCTATTCACCCAAGGAAACA1119    ValSerProAsnValCysHisSerAsnIleTyrSerProLysGluThr    250255260    ATCCCAGAAGAGGCACGAAGTGATATGCACTACAGTGTGGCTGAGGGC1167    IleProGluGluAlaArgSerAspMetHisTyrSerValAlaGluGly    265270275280    CTCAAACCTGCTGCCCCCTCAGCCCGAAATGCCCCCTACTTCCCTTGT1215    LeuLysProAlaAlaProSerAlaArgAsnAlaProTyrPheProCys    285290295    GACAAGGCCAGCAAAGAAGAAGAGAGACCCTCCTCGGAAGATGAGATT1263    AspLysAlaSerLysGluGluGluArgProSerSerGluAspGluIle    300305310    GCCCTGCATTTCGAGCCCCCCAATGCACCCCTGAACCGGAAGGGTCTG1311    AlaLeuHisPheGluProProAsnAlaProLeuAsnArgLysGlyLeu    315320325    GTTAGTCCACAGAGCCCCCAGAAATCTGACTGCCAGCCCAACTCGCCC1359    ValSerProGlnSerProGlnLysSerAspCysGlnProAsnSerPro    330335340    ACAGAGGCCTGCAGCAGTAAGAATGCCTGCATCCTCCAGGGTTCTGGC1407    ThrGluAlaCysSerSerLysAsnAlaCysIleLeuGlnGlySerGly    345350355360    TCCCCTCCAGCCAAGAGCCCCACTGACCCCAAAGCCTGCAGCTGGAAG1455    SerProProAlaLysSerProThrAspProLysAlaCysSerTrpLys    365370375    AAATACAAGTTCATCGTGCTCAACAGCCTCAACCAGAATGCCAAACCA1503    LysTyrLysPheIleValLeuAsnSerLeuAsnGlnAsnAlaLysPro    380385390    GGGGGGCCTGAGCAGGCTGAGCTGGGCCGCCTTTCCCCACGAGCCTAC1551    GlyGlyProGluGlnAlaGluLeuGlyArgLeuSerProArgAlaTyr    395400405    ACGGCCCCACCTGCCTGCCAGCCACCCATGGAGCCTGAGAACCTTGAC1599    ThrAlaProProAlaCysGlnProProMetGluProGluAsnLeuAsp    410415420    CTCCAGTCCCCAACCAAGCTGAGTGCCAGCGGGGAGGACTCCACCATC1647    LeuGlnSerProThrLysLeuSerAlaSerGlyGluAspSerThrIle    425430435440    CCACAAGCCAGCCGGCTCAATAACATCGTTAACAGGTCCATGACGGGC1695    ProGlnAlaSerArgLeuAsnAsnIleValAsnArgSerMetThrGly    445450455    TCTCCCCGCAGCAGCAGCGAGAGCCACTCACCACTCTACATGCACCCC1743    SerProArgSerSerSerGluSerHisSerProLeuTyrMetHisPro    460465470    CCGAAGTGCACGTCCTGCGGCTCTCAGTCCCCACAGCATGCAGAGATG1791    ProLysCysThrSerCysGlySerGlnSerProGlnHisAlaGluMet    475480485    TGCCTCCACACCGCTGGCCCCACGTTCGCTGAGGAGATGGGAGAGACC1839    CysLeuHisThrAlaGlyProThrPheAlaGluGluMetGlyGluThr    490495500    CAGTCTGAGTACTCAGATTCTAGCTGTGAGAACGGGGCCTTCTTCTGC1887    GlnSerGluTyrSerAspSerSerCysGluAsnGlyAlaPhePheCys    505510515520    AATGAGTGTGACTGCCGCTTCTCTGAGGAGGCCTCACTCAAGAGGCAC1935    AsnGluCysAspCysArgPheSerGluGluAlaSerLeuLysArgHis    525530535    ACGCTGCAGACCCACAGTGACAAACCCTACAAGTGTGACCGCTGCCAG1983    ThrLeuGlnThrHisSerAspLysProTyrLysCysAspArgCysGln    540545550    GCCTCCTTCCGCTACAAGGGCAACCTCGCCAGCCACAAGACCGTCCAT2031    AlaSerPheArgTyrLysGlyAsnLeuAlaSerHisLysThrValHis    555560565    ACCGGTGAGAAACCCTATCGTTGCAACATCTGTGGGGCCCAGTTCAAC2079    ThrGlyGluLysProTyrArgCysAsnIleCysGlyAlaGlnPheAsn    570575580    CGGCCAGCCAACCTGAAAACCCACACTCGAATTCACTCTGGAGAGAAG2127    ArgProAlaAsnLeuLysThrHisThrArgIleHisSerGlyGluLys    585590595600    CCCTACAAATGCGAAACCTGCGGAGCCAGATTTGTACAGGTGGCCCAC2175    ProTyrLysCysGluThrCysGlyAlaArgPheValGlnValAlaHis    605610615    CTCCGTGCCCATGTGCTTATCCACACTGGTGAGAAGCCCTATCCCTGT2223    LeuArgAlaHisValLeuIleHisThrGlyGluLysProTyrProCys    620625630    GAAATCTGTGGCACCCGTTTCCGGCACCTTCAGACTCTGAAGAGCCAC2271    GluIleCysGlyThrArgPheArgHisLeuGlnThrLeuLysSerHis    635640645    CTGCGAATCCACACAGGAGAGAAACCTTACCATTGTGAGAAGTGTAAC2319    LeuArgIleHisThrGlyGluLysProTyrHisCysGluLysCysAsn    650655660    CTGCATTTCCGTCACAAAAGCCAGCTGCGACTTCACTTGCGCCAGAAG2367    LeuHisPheArgHisLysSerGlnLeuArgLeuHisLeuArgGlnLys    665670675680    CATGGCGCCATCACCAACACCAAGGTGCAATACCGCGTGTCAGCCACT2415    HisGlyAlaIleThrAsnThrLysValGlnTyrArgValSerAlaThr    685690695    GACCTGCCTCCGGAGCTCCCCAAAGCCTGCTGAAGCATGGAGTGTTGATG2465    AspLeuProProGluLeuProLysAlaCys    700705    CTTTCGTCTCCAGCCCCTTCTCAGAATCTACCCAAAGGATACTGTAACACTTTACAATGT2525    TCATCCCATGATGTAGTGCCTCTTTCATCCACTAGTGCAAATCATAGCTGGGGGTTGGGG2585    GTGGTGGGGGTCGGGGCCTGGGGGACTGGGAGCCGCAGCAGCTCCCCCTCCCCCACTGCC2645    ATAAAACATTAAGAAAATCATATTGCTTCTTCTCCTATGTGNNNNNNNNNNNNNNNNNNN2705    NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN2765    NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN2825    NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN2885    NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN2945    NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN3005    NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN3065    NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN3125    NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN3185    NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN3245    NTTTAAGTATTGCATCTGTATAAGTAAGAAAATATTTTGTCTAAAATGCCTCAGTGTATT3305    TGTATTTTTTTGCAAGTGGGGGGTTACAATTTACCCAGTGTGTATTAAAAAAAACCCAAA3365    GAACCCAAAAATCTCCAGAAGGAAAAATGTGTAATTTTGTTCTAGTTTTCAGTTTGTATA3425    TACCCGTACAACGTGTCCTCACGGTGCCTTTTTTCACGGAAGTTTTCAATGATGGGCGAG3485    CGTGCACCATCCCTTTTTGAAGTGTAGGCAGACACAGGGACTTGAAGTTGTTACTAACTA3545    AACTCTCTTTGGGAATGTTTGTCTCATCCCANTCTGCGTCATGCTTGTGTGATAACTACT3605    CCGGAGACAGGGTTTGGCTGTGTCTAAACTGCATTACCGCGTTGTAAAAAATAGCTGTAC3665    CAATATAAGAATAAAATGTTGGAAAGTCGCAAAAAAAAAAAAAAAAAAAAAAAAA3720    (2) INFORMATION FOR SEQ ID NO:2:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 706 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:    MetAlaSerProAlaAspSerCysIleGlnPheThrArgHisAlaArg    151015    AspValLeuLeuAsnLeuAsnArgLeuArgSerArgAspIleLeuThr    202530    AspValValIleValValSerArgGluGlnPheArgAlaHisLysThr    354045    ValLeuMetAlaTrpArgGlyLeuPheTyrSerIlePheThrAspGln    505560    LeuLysCysAsnLeuSerValIleAsnLeuAspProGluIleAsnPro    65707580    GluGlyPheCysIleLeuLeuAspPheMetTyrThrSerArgLeuAsn    859095    LeuArgGluGlyAsnIleMetAlaValMetAlaThrAlaMetTyrLeu    100105110    GlnMetGluHisValValAspThrCysArgLysPheIleLysAlaSer    115120125    GluAlaGluMetValSerAlaIleLysProProArgGluGluPheLeu    130135140    AsnSerArgMetLeuMetProGlnAspIleMetAlaTyrArgGlyArg    145150155160    GluValValGluAsnAsnLeuProLeuArgSerAlaProGlyCysGlu    165170175    SerArgAlaPheAlaProSerLeuTyrSerGlyLeuSerThrProPro    180185190    AlaSerTyrSerMetTyrSerHisLeuProValSerSerLeuLeuPhe    195200205    SerAspGluGluPheArgAspValArgMetProValAlaAsnProPhe    210215220    ProLysGluArgAlaLeuProCysAspSerAlaArgProValProGly    225230235240    GluTyrSerArgProThrLeuGluValSerProAsnValCysHisSer    245250255    AsnIleTyrSerProLysGluThrIleProGluGluAlaArgSerAsp    260265270    MetHisTyrSerValAlaGluGlyLeuLysProAlaAlaProSerAla    275280285    ArgAsnAlaProTyrPheProCysAspLysAlaSerLysGluGluGlu    290295300    ArgProSerSerGluAspGluIleAlaLeuHisPheGluProProAsn    305310315320    AlaProLeuAsnArgLysGlyLeuValSerProGlnSerProGlnLys    325330335    SerAspCysGlnProAsnSerProThrGluAlaCysSerSerLysAsn    340345350    AlaCysIleLeuGlnGlySerGlySerProProAlaLysSerProThr    355360365    AspProLysAlaCysSerTrpLysLysTyrLysPheIleValLeuAsn    370375380    SerLeuAsnGlnAsnAlaLysProGlyGlyProGluGlnAlaGluLeu    385390395400    GlyArgLeuSerProArgAlaTyrThrAlaProProAlaCysGlnPro    405410415    ProMetGluProGluAsnLeuAspLeuGlnSerProThrLysLeuSer    420425430    AlaSerGlyGluAspSerThrIleProGlnAlaSerArgLeuAsnAsn    435440445    IleValAsnArgSerMetThrGlySerProArgSerSerSerGluSer    450455460    HisSerProLeuTyrMetHisProProLysCysThrSerCysGlySer    465470475480    GlnSerProGlnHisAlaGluMetCysLeuHisThrAlaGlyProThr    485490495    PheAlaGluGluMetGlyGluThrGlnSerGluTyrSerAspSerSer    500505510    CysGluAsnGlyAlaPhePheCysAsnGluCysAspCysArgPheSer    515520525    GluGluAlaSerLeuLysArgHisThrLeuGlnThrHisSerAspLys    530535540    ProTyrLysCysAspArgCysGlnAlaSerPheArgTyrLysGlyAsn    545550555560    LeuAlaSerHisLysThrValHisThrGlyGluLysProTyrArgCys    565570575    AsnIleCysGlyAlaGlnPheAsnArgProAlaAsnLeuLysThrHis    580585590    ThrArgIleHisSerGlyGluLysProTyrLysCysGluThrCysGly    595600605    AlaArgPheValGlnValAlaHisLeuArgAlaHisValLeuIleHis    610615620    ThrGlyGluLysProTyrProCysGluIleCysGlyThrArgPheArg    625630635640    HisLeuGlnThrLeuLysSerHisLeuArgIleHisThrGlyGluLys    645650655    ProTyrHisCysGluLysCysAsnLeuHisPheArgHisLysSerGln    660665670    LeuArgLeuHisLeuArgGlnLysHisGlyAlaIleThrAsnThrLys    675680685    ValGlnTyrArgValSerAlaThrAspLeuProProGluLeuProLys    690695700    AlaCys    705    (2) INFORMATION FOR SEQ ID NO:3:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 103 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:    AspGlySerPheValGlnHisSerValArgValLeuGlnGluLeuAsn    151015    LysGlnArgGluLysGlyGlnTyrCysAspAlaThrLeuAspValGly    202530    GlyLeuValPheLysAlaHisTrpSerValLeuAlaCysCysSerHis    354045    PhePheGlnSerLeuTyrGlyAspGlySerGlyGlySerValValLeu    505560    ProAlaGlyPheAlaGluIlePheGlyLeuLeuLeuAspPhePheTyr    65707580    ThrGlyHisLeuAlaLeuThrSerGlyAsnArgAspGlnValLeuLeu    859095    AlaAlaArgGluLeuArgVal    100    (2) INFORMATION FOR SEQ ID NO:4:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 107 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:    MetAspThrAlaSerHisSerLeuValLeuLeuGlnGlnLeuAsnMet    151015    GlnArgGluPheGlyPheLeuCysAspCysThrValAlaIleGlyAsp    202530    ValTyrPheLysAlaHisArgAlaValLeuAlaAlaPheSerAsnTyr    354045    PheLysMetIlePheIleHisGlnThrSerGluCysIleLysIleGln    505560    ProThrAspIleGlnProAspIlePheSerTyrLeuLeuHisIleMet    65707580    TyrThrGlyLysGlyProLysGlnIleValAspHisSerArgLeuGlu    859095    GluGlyIleArgPheLeuHisAlaAspTyrLeu    100105    (2) INFORMATION FOR SEQ ID NO:5:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 106 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:    MetAsnAsnSerSerGluLeuIleAlaValIleAsnGlyPheArgAsn    151015    SerGlyArgPheCysAspIleSerIleValIleAsnAspGluArgIle    202530    AsnAlaHisLysLeuIleLeuSerGlyAlaSerGluTyrPheSerIle    354045    LeuPheSerAsnAsnPheIleAspSerAsnGluTyrGluValAsnLeu    505560    SerHisLeuAspTyrGlnSerValAsnAspLeuIleAspTyrIleTyr    65707580    GlyIleProLeuSerLeuThrAsnAspAsnValLysTyrIleLeuSer    859095    ThrAlaAspPheLeuGlnIleGlySerAla    100105    (2) INFORMATION FOR SEQ ID NO:6:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 108 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:    CysLeuArgTrpAsnAsnHisGlnSerAsnLeuLeuSerValPheAsp    151015    GlnLeuLeuHisAlaGluThrPheThrAspValThrLeuAlaValGlu    202530    GlyGlnHisLeuLysAlaHisLysAsnValLeuSerAlaCysSerPro    354045    TyrPheAsnThrLeuPheValSerHisProGluLysHisProIleVal    505560    IleLeuLysAspValProTyrSerAspMetLysSerLeuLeuAspPhe    65707580    MetTyrArgGlyGluValSerValAspGlnGluArgLeuThrAlaPhe    859095    LeuArgValAlaGluSerLeuArgIleLysGlyLeu    100105    (2) INFORMATION FOR SEQ ID NO:7:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 109 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:    GlnTyrSerAsnGluGlnHisThrAlaArgSerPheAspAlaMetAsn    151015    GluMetArgLysGlnLysGlnLeuCysAspValIleLeuValAlaAsp    202530    AspValGluIleHisAlaHisArgMetValLeuAlaSerCysSerPro    354045    TyrPheTyrAlaMetPheThrSerPheGluGluSerArgGlnAlaArg    505560    IleThrLeuGlnSerValAspAlaArgAlaLeuGluLeuLeuIleAsp    65707580    TyrValTyrThrAlaThrValGluValAsnGluAspAsnValGlnVal    859095    LeuLeuThrAlaAlaAsnLeuLeuGlnLeuThrAspVal    100105    (2) INFORMATION FOR SEQ ID NO:8:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 105 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:    GlnLeuGlnAsnProSerHisProThrGlyLeuLeuCysLysAlaAsn    151015    GlnMetArgLeuAlaGlyThrLeuCysAspValValIleMetValAsp    202530    SerGlnGluPheHisAlaHisArgThrValLeuAlaCysThrSerLys    354045    MetPheGluIleLeuPheHisArgAsnSerGlnHisTyrThrLeuAsp    505560    PheLeuSerProLysThrPheGlnGlnIleLeuGluTyrAlaTyrThr    65707580    AlaThrLeuGlnAlaLysAlaGluAspLeuAspAspLeuLeuTyrAla    859095    AlaGluIleLeuGluIleGluTyrLeu    100105    (2) INFORMATION FOR SEQ ID NO:9:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 110 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:    CysLeuGlnPheThrArgHisAlaSerAspValLeuLeuAsnLeuAsn    151015    ArgLeuArgSerArgAspIleLeuThrAspValValIleValValSer    202530    ArgGluGlnPheArgAlaHisLysThrValLeuMetAlaCysSerGly    354045    LeuPheTyrSerIlePheThrAspGlnLeuLysCysAsnLeuSerVal    505560    IleAsnLeuAspProGluIleAsnProGluGlyPheCysIleLeuLeu    65707580    AspPheMetTyrThrSerArgLeuAsnLeuArgGluGlyAsnIleMet    859095    AlaValMetAlaThrAlaMetTyrLeuGlnMetGluHisVal    100105110    __________________________________________________________________________

What is claimed is:
 1. A method for detecting a rearrangment of a bcl-6gene in B-cell lymphoma in a subject, comprising:(a) obtaining a DNAsample from the subject; (b) cleaving the DNA sample into fragments; (c)seperating the DNA fragments by size fractionation; (d) hybidizing theDNA fragments with a nucleic acid molecule comprising at least 15contiguous nucleotides which is complementary to a sequence of anisolated nucleic acid molecule having the nucleic acid sequence as setforth in SEQ ID NO:1 and specifically hybridizes with the nucleic acidsequence as set forth in SEQ ID NO:1 to detect the DNA fragmentcontaining the bcl-6 nucleic acid sequence; and (e) comparing thedetected DNA fragment from (d) with a DNA fragment from a known normalsubject, the difference in size of the fragments indicating occurrenceof a rearrangement of the bcl-6 gene in B-cell lymphoma in the subject.2. The method of claim 1, where in step (b), the DNA sample is cleavedby restriction enzyme.
 3. The method of claim 1, wherein the sizefractionation is step (c) is effected by a polyacrylamide or agarosegel.
 4. The method of claim 1, where in step (d), the nucleic acidmolecule is labeled with a detectable marker.
 5. The method of claim 4,wherein the detectable marker is a radiolabelled molecule, a fluorescentmolecule, an enzyme, or a ligand.
 6. The method of claim 1, furthercomprising transferring the DNA fragments into a solid matrix beforestep (d).
 7. A method for detecting a rearrangement of a bcl-6 gene inB-cell lymphoma in a subject, comprising:(a) obtaining an RNA samplefrom the subject; (b) separating the RNA sample into different speciesby size fractionation; (c) hybridizing the RNA species with a nucleicacid molecule comprising at least 15 contiguous nucleotides which iscomplementary to a sequence of an isolated nucleic acid molecule havingthe nucleic acid sequence as set forth in SEQ ID NO:1 and specificallyhybridizes with the nucleic acid sequence as set forth in SEQ ID NO:1 todetect the RNA species containing bcl-6 nucleic acid sequence; and (d)comparing the detected RNA species from step (c) with the RNA speciesfrom a known normal subject, the difference in size of the speciesindicating the rearrangement of the bcl-6 gene in B-cell lymphoma in thesubject.
 8. The method of claim 7, wherein the size fractionation instep (b) is effected by a polyacrylamide or agarose gel.
 9. The methodof claim 7, where in step (c), the nucleic acid molecule is labeled witha detectable marker.
 10. The method of claim 9, wherein the detectablemarker is a radiolabelled molecule, a fluorescent molecule, an enzyme,or a ligand.
 11. A method of claim 7, further comprising transferringthe RNA species into a solid matrix before step (c).